# Characterization of the Tunisian Phosphate Rock from Metlaoui-Gafsa Basin and Bio-Leaching Assays

Boutheina Messai, Ines Taieb, Sonia Ben Younes, Bruno Lartiges, Ezzedine Ben Salem, Ali Ellafi

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Boutheina Messai, Ines Taieb, Sonia Ben Younes, Bruno Lartiges, Ezzedine Ben Salem, et al.. Characterization of the Tunisian Phosphate Rock from Metlaoui-Gafsa Basin and Bio-Leaching Assays. Sustainability, 2023, 15 (9), pp.7204. 10.3390/su15097204. hal-04931899

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Article

# Characterization of the Tunisian Phosphate Rock from Metlaoui-Gafsa Basin and Bio-Leaching Assays

Boutheina Messai<fnref n="1" />,<fnref n="2" />, Ines Taieb<fnref n="2" />,<fnref n="3" />, Sonia Ben Younes<fnref n="2" />,<fnref n="4" />,* ID, Bruno Lartiges<fnref n="5" />, Ezzedine Ben Salem<fnref n="1" /> and Ali Ellafi<fnref n="2" />,<fnref n="3" />,* ID

<figure><img src="image_2.png" /></figure>

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**Citation:** Messai, B.; Taieb, I.; Ben Younes, S.; Lartiges, B.; Ben Salem, E.; Ellafi, A. Characterization of the Tunisian Phosphate Rock from Metlaoui-Gafsa Basin and Bio-Leaching Assays. *Sustainability* **2023**, *15*, 7204. https://doi.org/10.3390/su15097204

Academic Editors: Hedi Ben Mansour and Asma Beltifa

Received: 24 December 2022

Revised: 5 February 2023

Accepted: 24 February 2023

Published: 26 April 2023

<figure><img src="image_3.png" /></figure>

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

**Abstract:** Soil contamination by heavy metals through the application of a phosphate fertilizer is a key issue for sustainable agriculture. Among contaminants, cadmium (Cd) is considered the most hazardous to human beings' health and the surrounding environment. X-ray diffraction (XRD), combined with binocular mineralogical analysis and chemical analysis, was used to determine the C(I) and C(II) layers' composition. In the C(II) (+71 µm)-size fraction, the presence of carbonate-fluorapatite, illite, and montmorillonite was revealed, whereas in the C(I) (–71 µm)-size fraction, carbonate-fluorapatite, calcite, quartz, sanidine, clinoptilolite, and taramovite were identified. The P₂O₅ and CaO contents were greater in the C(II) layer than that in the C(I) layer, whereas SiO₂, MgO, CO₂, Cd, Zn, and CO₃ were higher in the C(I) layer than that in the C(II) layer. The bioleaching of Cd from phosphate rock samples from the Kef Eddour deposit was investigated using three bacterial strains isolated from the local phosphate environment. A decrease in cell viability was noticed when the phosphate C(I) and C(II) samples showed toxicity in the samples. In addition, the isolated bacteria, which were initially moderately hydrophobic, changed to strongly hydrophobic. The use of the consortium (S1 + S2 + S3) was the most powerful combination to reduce the Cd content, which increased from 13.31% to 29.07% using S3 and the consortium (S1 + S2 + S3), respectively, when the C(II) (+71 µm)-size fraction sample was added to the medium. The same result was shown when the C(I) (–71 µm)-size fraction sample was used. The isolated strains could be used as a biological tool for bioleaching.

**Keywords:** phosphate rock; bioleaching; bacteria; Kef Eddour

## 1. Introduction

The Tunisian phosphate industry and its derivative sectors are an important part of the national economy, contributing to 8% of the Tunisian gross national product [1]. Phosphate mining has operated in the Gafsa basin for more than a century. The current production capacity has reached 8.5 million tons in Gafsa through 8 open-pit mines. All production was intended for export until the early 1970s. Two years later, Tunisia gradually acquired phosphate transformation and manufacturing units to produce fertilizers that are distributed in the form of chemical complexes [2].

Consequently, Tunisia was ranked fifth worldwide among the producing countries in 2010. More precisely, the Metlaoui-Kef Eddour ores provide pure phases of about 31% P₂O₅

<footnote n="1">¹ Unit of Materials and Organic Synthesis UR17ES31, Preparatory Institute for Engineering Studies of Monastir, University of Monastir, Monastir 5000, Tunisia</footnote>

<footnote n="2">² Faculty of Sciences of Gafsa, Campus Universitaire Sidi Ahmed Zarroug, University of Gafsa, Gafsa 2112, Tunisia</footnote>

<footnote n="3">³ Laboratory of Analysis, Treatment and Valorization of the Pollutants of the Environment and Products, Faculty of Pharmacy, University of Monastir, Monastir 5000, Tunisia</footnote>

<footnote n="4">⁴ Research Unit n°17/ES/13, Faculty of Medicine of Tunis, University of Tunis El Manar, Tunis 1007, Tunisia</footnote>

<footnote n="5">⁵ Laboratory of Analysis, Geosciences Environment Toulouse, University of Toulouse III (Paul Sabatier), 31400 Toulouse, France</footnote>

<footnote>* Correspondence: sonia.benyounes@fsgf.ugaf.tn (S.B.Y.); ali_lafi160@yahoo.fr (A.E.); Tel.: +216-76-21-10-24 (S.B.Y.); +216-73-466-244 (A.E.); Fax: +216-76-21-10-26 (S.B.Y.); +216-73-461-830 (A.E.)</footnote>
and a bone phosphate lime concentration equal to 65–67%. High-quality concentrations of triple phosphate (70–73%) (ore grade of approximately 62% calcium triphosphate) are called “Extra” thanks to substitutions in the crystal lattice of the francolite, which makes Tunisian phosphates the most soluble in the world, even more so than North Carolina phosphates. However, due to international constraints concerning the export of phosphate-containing fertilizers, especially the strict standards regarding Cd content, phosphate fertilizers that are derived from Gafsa ores do not meet the Cd limit values adopted by the European Union. This limitation of the Cd content has a major effect on the marketability of these raw fertilizer minerals [3].

Cadmium is not an essential element and is of very high toxicity to plants and human health [4–6]. It is generally introduced in agricultural soils through the application of phosphate fertilizers [7]. Several methods can be used to reduce the cadmium content in phosphate fertilizers during phosphate rock processing, such as chemical methods based on flushing and electrokinetic extraction, in addition to immobilization technology, with a reduction in permeability and solubility [8] and, recently, phytoremediation using hyperaccumulator plants [6,9]. Moreover, guttering enables the removal of unwanted impurities by applying a rapid heat treatment in an infrared oven followed by a chemical treatment [10]. However, those methods have been proven to be rather inefficient and very expensive for the corresponding limited improvements in biological activities and fertility [6]. Bioleaching has also been responsible for reducing Cd contamination in phosphate rocks [6,11]. It has been shown to be an efficient, sustainable, and cost-effective method that allows Cd to be eliminated without generating new pollution [6,12]. Recently, the removal of environmental toxic waste via biological agents, named bioremediation, has become an innovative, effective tool that could be the key to addressing this problem [6,13]. Microorganisms deploy many mechanisms for their survival in and resistance to metallic environments, including metal oxidation, methylation, enzymatic degradation, metal-organic complexion, decreasing the metal concentration, metal-ligand degradation, metal efflux pumps, demethylation, intracellular and extracellular metal sequestration, exclusion by a permeability barrier, and production of metal chelators such as metallothioneins and biosurfactants. For example, to eliminate metallic contaminants, microorganisms can utilize valence conversion, volatilization, or extracellular chemical precipitation [13].

In this survey, the chemical characterization of phosphate rock from Kef Eddour Metlaoui-Gafsa was investigated. In addition, after the bioleaching assays, three bacteria were characterized in terms of cell viability, biofilm formation, and hydrophobicity. The reduction in cadmium content in the presence of phosphate from the C(I) and C(II) layers was assayed.

## 2. Materials and Methods

### 2.1. *Sampling and Characterization of the Phosphate Rock*

#### 2.1.1. Sampling

Samples were collected from the phosphate rock from the Metlaoui-Kef Eddour Center open-pit ores (Gafsa Basin, Tunisia) in January 2018 (Figure 1). The Kef Eddour Center deposit consists of eight phosphate rock layers, referred to as C(I), C(II), C(III+IV), C(V), C(VI), C(VII), and C(VIII). Stratigraphic information for the Kef Eddour center deposit is shown in Table 1. Samples from layers C(I) and C(II) were selected for their high Cd levels (Table 1). The usual Cd concentration of those layers ranges from 31 to 82 ppm and from 32 to 67 ppm for layers C(I) and C(II), respectively. Each phosphate layer was scraped with a hammer to a depth of about 5 to 10 cm in order to remove any rock altered by external agents such as wind and precipitation. Fifty kilograms of phosphate rock was then collected from each layer. Eight samples were taken from the Kef Eddour center section and stored in plastic bags. Samples from each layer were then homogenized, quartered, and dried in an oven at 105 °C for 24 h, while the remaining phosphate rock was stored. The fraction sizes of <2 mm–71 µm (i.e., the merchant phosphate) and <71 µm (phosphate
sludge), referred to as +71 µm and −71 µm of the dried phosphate rock, were separated using ASTM sieves.

<table><caption>**Table 1.** Stratigraphic information of the Kef Eddour center (1/200 (CPG)) (modified).</caption><thead><tr><th>LOG</th><th>Power Ratings</th><th>Layers</th><th>Descriptions</th></tr></thead><tbody><tr><td></td><td>4.56</td><td>C(I)</td><td>Pelphospharenite, coprolite, bioclastic, very slightly lithoclastic</td></tr><tr><td></td><td>0.95</td><td>m 1-2</td><td>Olive-green marl</td></tr><tr><td></td><td>2.40</td><td>C(II)</td><td>Homogeneous phospharenite, rich in tricalcium phosphate, carbonate</td></tr><tr><td></td><td>0.82</td><td>m 2-3</td><td>Greenish marls</td></tr><tr><td></td><td>0.40</td><td>II</td><td>Pelphospharenite very marly, grey</td></tr><tr><td></td><td>0.33</td><td>m 3-4</td><td>Greenish grey marls</td></tr><tr><td></td><td>0.23</td><td>C(IV) a</td><td>Coarse, carbonated pelphospharenite</td></tr><tr><td></td><td>0.37</td><td>m 4a-b</td><td></td></tr><tr><td></td><td>0.38</td><td>C(IV) b</td><td>Grey pelphospharenite, very hard, very coprolite</td></tr><tr><td></td><td>4.13</td><td>m 4-5</td><td>Greenish grey marls, carbonate banks, very hard whitish ones</td></tr><tr><td></td><td>1.06</td><td>C(V)</td><td>Grey Pelphospharenite</td></tr><tr><td></td><td>0.73</td><td>m 5-6</td><td>Greenish, hard marls</td></tr><tr><td></td><td>1.27</td><td>C(VI)</td><td>Grey Pelphospharenite</td></tr><tr><td></td><td>3.60</td><td>m 6-7a</td><td>Cherts greyish white</td></tr><tr><td></td><td>0.43</td><td>C(VII) a</td><td>Carbonate, magnesian</td></tr><tr><td></td><td>0.72</td><td>m 7a-b</td><td>Greenish-grey marl limestone</td></tr><tr><td></td><td>0.76</td><td>C(VII) b</td><td>Grey pelphospharenites, not very marly</td></tr><tr><td></td><td>0.74</td><td>m 7b-8</td><td>Continuous carbonate benches, very hard blankets</td></tr><tr><td></td><td>1.19</td><td>C(VIII)</td><td>Soft, bioclastic pelphospharenite</td></tr><tr><td></td><td></td><td></td><td>Base shell</td></tr></tbody></table>

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<figure><img src="image_5.png" /><figcaption>**Figure 1.** Kef Eddour center geographic location from Google earth.</figcaption></figure>

### 2.1.2. Characterization of Phosphate Rock

The identification of phosphate particles was carried out by optical microscopy (RE-LEAX FRANCE binocular magnifier). The elemental analysis of the samples was conducted by flame atomic absorption spectroscopy for the Cd, Zn, Mg and Si determination contents and by light absorption spectrophotometry for the P and Ca determination contents. The X-ray powder diffraction (XRD) analysis was performed at room temperature using a "Philips MPD1880-PW1710" diffractometer using a CuKα radiation (λ = 0.15418 nm) in the 2–80° range with a 0.02° pitch and a counting time of 20 s/step. The mineral phase identification was determined using International Centre for Diffraction Data (ICDD) standard cards. The phosphate samples were also observed with a scanning electron microscope (Jeol JSM 6363LV, Tokyo, Japan) equipped with a silicon drift detector (SDD) PGT operating at 20 kV for microanalysis. The particles were placed on double-sided adhesive conductive carbon tape and coated with carbon [14].

## 2.2. *Bio-Treatment of the Phosphate Rock*

### 2.2.1. The Used Bacteria

In this study, three strains (S1, S2, and S3) were isolated using Luria Bertani (LB) medium supplemented with the autoclaved effluent (100%) from Metlaoui phosphate laundries discharged in Oued sakdoud, Gafsa Governorate, South-West of Tunisia (north latitude 34°20′, east longitude 8°22′). The strain S3 was identified as *Lysinibacillus fusiformis* strain ZC (ID: KX148607) [13]. The isolated strains were inoculated on LB broth (LB). After incubation at 37 °C for 24 h, various tests were performed to identify the isolated strains (Gram staining, catalase test, and oxidase test).

### 2.2.2. Bioleaching In Vitro Assay

The bioleaching assays were performed using two grams of the phosphate samples (C(II) and C(I)) dissolved in 100 mL of acidified water (distilled water (90%) + concentrated
nitric acid (10%)). Each microcosm containing the phosphate sample was inoculated with the isolated bacteria ($10^9$ CFU/mL) and incubated at 37 °C for 24–48 h.

### 2.2.3. Cell Viability Essay

A cell viability essay was performed using a colorimetric assay from yellow to violet [13]. The reduction of tetrazolium salt (MTT) to soluble formazon salt water was the result of the metabolically active cells. The experimental protocol was carried out as described by Taieb et al. (2021) [13]. Finally, the produced solubilized formazon was quantified by photometry at 570 nm. Viability was determined in each microcosm at the time of inoculation (0 h), after 24 h, and after 48 h [13]. To calculate the reduction in viability compared with that in the negative control, the following equation was used: $% viab = 100 \times (OD_{570s}/OD_{570c})$.

### 2.2.4. Biofilm Formation Assays

#### Qualitative Biofilm Study

The isolated bacteria biofilm formation was qualitatively detected, in triplicate, on Congo red agar (CRA) plates [13]. These inoculated CRAs were incubated for 24 h at 37 °C and overnight in the dark at ambient temperature. Slime-producing bacteria appeared as black colonies, whereas non-slime producers remained unpigmented [13].

#### Characterization of Semi-Quantitative Slime-Producing Bacteria

Biofilm production by isolated bacteria was determined using a semi-quantitative adherence assay on 96-well tissue culture plates [13] without any modification. The optical density (OD) of each well was measured at 570 nm ($OD_{570}$) using an ELISA reader (automated multiskan reader: MNOP, Rome, Italy). Biofilm formation was interpreted as highly positive ($OD_{570} \ge 1$), low-grade positive ($0.1 \le OD_{570} < 1$), or negative ($OD_{570} < 0.1$) [13,15].

### 2.2.5. Hydrophobicity Study

Hydrophobicity was measured using the hexadecane method [13]. The bacterial cells, grown overnight in LB, were washed with PBS and re-suspended in 4 mL of PBS, and the absorbance ($OD_{540}$) was determined. One milliliter of hexadecane (Sigma-Aldrich, Waltham, MA, USA) was added to each cell suspension, incubated for 10 min, and re-incubated at 37 °C for 30 min. The aqueous layer was removed and aerated to remove all traces of hexadecane, and absorbance ($OD_{540}$) was measured using a UV spectrophotometer (UV-1800 Shimadzu, Kyoto, Japan) against a hexadecane-extracted PBS blank. The hydrophobicity index was expressed as the ratio of absorbance of the hexadecane-extracted sample to absorbance of the sample before extraction. Each experiment was performed in triplicate. The bacterial cells were classified as strongly hydrophobic, moderately hydrophobic, moderately hydrophilic, and hydrophilic when the number of cells bound to hexadecane was higher than 55%, between 30 and 54%, between 10 and 29%, and less than 10%, respectively [16].

### 2.2.6. Atomic Absorption Spectroscopy

The metal-processing ability of the three isolated bacteria (S1, S2 and S3) were evaluated by evaluating the Cd amount variation in the supernatant via atomic absorption spectrophotometer [13]. For each strain, two microcosms were used for bacterial growth under cadmium stress (phosphate rock addition). A microcosm without bacteria was used as the control. Microcosms were placed in a shaking incubator at 37 °C. After 24 h, 5 mL aliquots were taken from each microcosms and the cell culture was centrifuged at 6000 rpm for 10 min. The supernatants were used for Cd level measurement.

## 2.3. Statistical Analysis

Statistical analysis was performed using the SPSS 13.0 statistics package for Windows. $p$-values < 0.05 were considered as significant. All experiments were performed in triplicate.
## 3. Results

### 3.1. Characterization of Phosphate Size Fractions

#### 3.1.1. X-ray Powder Diffraction Analysis

An X-ray diffraction analysis of the phosphate fractions of the Kef Eddour center C(II) (+71 µm) and that of C(I) (–71 µm) were determined (Figure 2). In the C(II) (+71 µm)-size fraction, the analysis revealed the presence of carbonate-fluorapatite, illite, and montmorillonite. The number of phosphate phases was calculated using the Rietveld method, and it was about 97.9 %, which was confirmed by binocular observations (~95%) (Figure 2a). However, in the C(I) (–71 µm)-size fraction (layer I) (Figure 2b), the results proved the presence of carbonate-fluorapatite, calcite, quartz, sanidine, clinoptilolite, and taramovite. Therefore, the X-ray diffraction of the Tunisian phosphate rocks are similar to that of other phosphate rocks from Morocco and Algeria [17,18].

<figure><img src="image_6.png" /><figcaption>**Figure 2.** XRD pattern of (a) C(II) (+71 µm)-size fraction of phosphate Kef Eddour center (layer II). (b) C(I) (–71 µm) (sludge) of phosphate Kef Eddour center (layer I).</figcaption></figure>

#### 3.1.2. Chemical Analysis

The major and trace elements of the phosphate samples were chemically analyzed to identify the potential environmental damage that phosphate wastes may cause. The phosphate ores were divided into three groups based on their P₂O₅ content: low-grade ores (12–16%), medium-grade ores (17–25%), and high-grade ores (26–35%). Deposits that are mined and processed must contain about 28–38% P₂O₅ to be accepted as economically commercial phosphate [19]. The P₂O₅ and CaO contents were greater in the C(II) layer than in the C(I) layer, whereas the SiO₂, MgO, CO₂, Cd, Zn, and COrg contents were higher in the C(I) layer than in the C(II) layer. The results are listed in Table 2. These results are in accordance with the chemical analyses of merchant phosphates from Algeria, Morocco, and Tunisia, as stated before [20–22]. The Cd content, a toxic element found in both the C(I) and C(II) layers, varied between 67.23 and 81.30 ppm. In fact, the C(I) layer was richer in Cd than the C(II) layer. This Cd content is one of the main factors which determine the phosphate resource quality [23].
<table><caption>**Table 2.** Chemical analysis of phosphate samples.</caption><thead><tr><th>Layers</th><th>Meshes</th><th>Weight (g)</th><th>Weight Yields%</th><th>P<sub>2</sub>O<sub>5</sub> %</th><th>CaO %</th><th>SiO<sub>2</sub> %</th><th>MgO %</th><th>CO<sub>2</sub> %</th><th>CO<sub>rg</sub> %</th><th>Cd ppm</th><th>Zn ppm</th></tr></thead><tbody><tr><td>C(I)</td><td>−71 µm</td><td>281 ± 12</td><td>13.59 ± 0.023</td><td>11.30 ± 1.73</td><td>22.98 ± 1.49</td><td>24.10 ± 0.98</td><td>1.69 ± 0.07</td><td>13.10 ± 1.06</td><td>3.31 ± 0.57</td><td>81.38 ± 4.75</td><td>572 ± 19.33</td></tr><tr><td>C(II)</td><td>+71 µm</td><td>1670 ± 24.90</td><td>67.26 ± 4.51</td><td>29.09 ± 2.13</td><td>45.83 ± 2.15</td><td>4.97 ± 0.83</td><td>0.59 ± 0.08</td><td>10.08 ± 1.37</td><td>2.85 ± 0.96</td><td>67.23 ± 3.72</td><td>500 ± 24.21</td></tr></tbody></table>

### 3.1.3. Electron Microscopy

The examination of the C(I) (−71 µm) and C(II) (+71 µm) samples by scanning electron microscopy (SEM) coupled with an energy-dispersive X-ray spectroscopy (EDXS) analysis allowed for the characterization of the phosphate rock (Figure 3). The C(II) (+71 µm) sample showed the predominance of the rounded particles of carbonated fluoroapatite, with a few occurrences of carbonates and Cd-bearing sphalerite particles (Zn/Cd elemental ratio ranging from 9.3 to 10). The phosphate particles contained micron-size pyrite particles and, more rarely, Cd-bearing sphalerite particles (Zn/Cd elemental ratio ranging from 14 to 16). Manual sorting of the phosphate particles, under a binocular microscope, gave an estimation of 94% phosphate mineral phases, 5% carbonate particles and 1% quartz particles. The C(I) (−71 µm) sample revealed abundant micron-size clay particles that enmeshed a few remaining phosphate and carbonate larger particles. Framboidal pyrite and Cd-bearing particles were also found in the sample.

<figure><img src="image_7.png" /><figcaption>**Figure 3.** Micrographs in the back-scattered electron mode of merchant phosphate C(II) (+71 µm)-size fraction (a,b) and of phosphate sludge C(I) (−71 µm)-size fraction (c,d). A: carbonated fluoroapatite; Py: pyrite; C: carbonate; ZnS (Cd): Cd-bearing sphalerite.</figcaption></figure>
## 3.2. Bioleaching Procedure

### 3.2.1. Characterization of the Newly Isolated Bacteria

The three isolated bacteria were mesophilic. The three strains were rod-shaped (bacilli). Another criterion, called the Gram stain, was used to distinguish and classify bacterial species into Gram-positive (Gram+) and Gram-negative (Gram−) groups by detecting the presence or absence of peptidoglycan in the bacterial cell wall [24]. All the isolates were Gram+, which were stained purple. The oxidoreductase activity was also determined. The S1 strain was catalase-positive, and the S2 and S3 stains were catalase-negative. The oxidase test proved that all strains are not dotted by this oxidoreductase activity (Table 3). The Tunisian biotopes were rich in microorganisms, especially bacteria dotted with many activities [24–26].

<table><caption>**Table 3.** Biochemical characterization of the isolated bacteria.</caption><thead><tr><td>Strains</td><td>Isolation Temperature</td><td>Shape</td><td>Gram Staining</td><td>Catalase</td><td>Oxidase</td></tr></thead><tbody><tr><td>S1</td><td>30 °C</td><td>Bacile</td><td>Gram+</td><td>+</td><td>—</td></tr><tr><td>S2</td><td>30 °C</td><td>Bacile</td><td>Gram+</td><td>—</td><td>—</td></tr><tr><td>S3</td><td>30 °C</td><td>Bacile</td><td>Gram+</td><td>—</td><td>—</td></tr></tbody></table>

### 3.2.2. Cell Viability

The results of the cell viability essay are presented in Figure 4 using the three isolated strains incubated in the presence of a phosphate rock in different microcosms. The results show a decrease in the percentage of cell viability after incubation with C(II) (+71 µm) and C(I) (−71 µm) (Figure 4), but the isolated bacteria remained viable even after 48 h of incubation. From the first day of incubation, a slight decrease (from 5 to 45%) in the bacteria number was observed in all microcosms and regardless of the origin of the phosphate sample. Thus, in the case of the C(I) (−71 µm) layer, the cell viability was different from the control and decreased more than in the case of the C(II) (+71 µm) layer after 48 h of incubation. In fact, this test reflects the ability of the bacteria to survive in a hostile environment rich in Cd. This Cd level reduction may be due to various mechanisms. Thus, the isolated strains use resistance mechanisms to cope with the Cd-induced metal stress present at various levels in the phosphate. Cd-resistant strains use a number of strategies for their survival, such as the consumption of trace amounts of toxic metal ions in their metabolism and detoxification of excess metal [27].

<figure><img src="image_8.png" /><figcaption>**Figure 4.** Cell viability of the three isolated strains S1 (■), S2 (■) and S3 (■) in the absence and the presence of phosphate merchant C(II) (+71 µm) and C(I) (−71 µm) after incubation at 0 h, 24 h and 48 h (■).</figcaption></figure>
### 3.2.3. Biofilm Formation

The quantitative tests revealed that a biofilm formation (0.1 < OD570nm) in the presence of phosphate particles compared with the control was observed (Table 4). The values show that strain S3 is the most productive of the biofilms in the cases of the C(I) and C(II) layers in comparison with the control. In addition, biofilm formation was higher in S2 and S3 in the case of the C(I) (−71 µm) layer, which shows high toxicity due to Cd. In the present work, a correlation between qualitative and quantitative biofilm formation was identified. Indeed, under stress, the selected strains grow a biofilm to survive, to adapt to, and to resist Cd-contaminated conditions [28]. Thus, the formation of biofilms is one of the adaptive mechanisms used by bacteria to escape harsh conditions, such as metallic contaminants, which may cause alterations in the structure and functions of microbial communities. Moreover, it has been proven that slime production plays an important role in the resistance of various microorganisms [29].

<table><caption>**Table 4.** Biofilm formation of the three isolated strains in the presence of the phosphate C(II) (+71 µm) and C(II) (+71 µm) layers.</caption><thead><tr><th rowspan="2">Strains</th><th colspan="3">Control</th><th colspan="3">C(II) (+71 µm)</th><th colspan="3">C(I) (−71 µm)</th></tr><tr><th>S1</th><th>S2</th><th>S3</th><th>S1</th><th>S2</th><th>S3</th><th>S1</th><th>S2</th><th>S3</th></tr></thead><tbody><tr><td>OD<sub>570</sub></td><td>0.04 ±<br>0.003</td><td>0.04 ±<br>0.002</td><td>0.04 ±<br>0.003</td><td>0.10 ±<br>0.004</td><td>0.24 ±<br>0.009</td><td>0.30 ±<br>0.012</td><td>0.1 ±<br>0.0017</td><td>0.21 ±<br>0.009</td><td>0.35 ±<br>0.011</td></tr></tbody></table>

### 3.3. Cell Surface Hydrophobicity

Hydrophobicity was measured using the hexadecane method. The bacterial cell adhesion to hexadecane proved that the three studied strains can be considered hydrophobic under stressful conditions (Table 5). In fact, the obtained results indicated that strain S1 is hydrophilic, whereas S2 and S3 are moderately hydrophobic in the absence of cadmium stress. In the case of the phosphate C(I) and C(II) layers, the three strains changed to strongly hydrophobic strains, showing the effect of the phosphate-rich metal aggressor (Table 5). Thus, for strain S3, the hydrophobicity increased from 42% to 82% in the control and to 86% in the C(II) (+71 µm) and C(I) (−71 µm) layers. Such increased hydrophobicity is often related to an enhanced biofilm growth [28]. In fact, the hydrophobicity increased from moderately to strongly hydrophobic in the phosphate samples. In the case of microorganisms, hydrophobicity is considered as a virulence factor that interferes with the adhesion of microorganisms to biotic and abiotic surfaces. This hydrophobicity variation is due to the composition of the phosphate layers, which are rich in metals. In fact, the surface properties of stressed strains are different. Considering that hydrophobicity is the most important factor that is expected to contribute to survival in adverse conditions. Generally, changes in the cell surface properties of bacteria play an important role in its resistance to severe conditions and in the removal of contaminants [13].

<table><caption>**Table 5.** Effect of the presence of the phosphate C(II) (+71 µm) and C(I) (−71 µm) layers on surface hydrophobicity by the three isolated bacteria.</caption><thead><tr><th>Strains</th><th>Control</th><th>C(II) (+71 µm)</th><th>C(I) (−71 µm)</th></tr></thead><tbody><tr><td>S1</td><td>28 ± 2.82%</td><td>69 ± 2.49%</td><td>73 ± 2.07%</td></tr><tr><td>S2</td><td>39 ± 1.77%</td><td>66 ± 1.95%</td><td>77 ± 1.45%</td></tr><tr><td>S3</td><td>42 ± 2.80%</td><td>82 ± 2.43%</td><td>86 ± 2.64%</td></tr></tbody></table>
### 3.4. Effect of Bacterial Treatment on Soluble Cd Content

The soluble Cd content after incubation of microcosms under various additions of pure or mixed bacterial strains is shown in Table 6. A significant decrease in Cd level is found in comparison with the control. Using each strain alone, S3 is the most efficient at reducing Cd content in the supernatants in the cases of both the C(I) and C(II) layers. This result is not surprising since strain S3, identified as *Lysinibacillus fusiformis* strain ZC, can accumulate and/or adsorb metal ions such as Cu, Zn, Cd, Mg, and Pb. In fact, the percentage of accumulation and/or adsorption does not exceed 4.6% for Cd at a concentration of 1 mM [13]. The combination S2 + S3 is more efficient at decreasing the soluble Cd content. However, the use of the consortium (S1 + S2 + S3) is the most powerful combination for reducing the Cd content. The Cd concentration increased from 13.31% to 29.07% using S3 and the consortium (S1 + S2 + S3) when the C(II) (+71 µm)-size fraction sample was added to the medium. The same result was shown when the C(I) (–71 µm)-size fraction sample was added to the medium, with an increase in percentage varying from 13.12% to 31.61% using S3 and the consortium (S1 + S2 + S3), respectively. These results confirm that the strains are able to adsorb or absorb Cd.

<table><caption>**Table 6.** Cd concentration after bioleaching assays using atomic absorption spectrometry (AAS) technique.</caption><thead><tr><th rowspan="2">Strains</th><th colspan="2">Cd Content (ppm)</th></tr><tr><th>C(II) (+71 µm)</th><th>C(I) (–71 µm)</th></tr></thead><tbody><tr><td>Control</td><td>67.23 ± 8.12</td><td>81.30 ± 9.94</td></tr><tr><td>S<sub>1</sub></td><td>59.78 ± 7.75</td><td>73.21 ± 9.49</td></tr><tr><td>S<sub>2</sub></td><td>61.68 ± 6.30</td><td>72.60 ± 7.72</td></tr><tr><td>S<sub>3</sub></td><td>58.28 ± 7.13</td><td>70.63 ± 8.73</td></tr><tr><td>S<sub>1</sub> + S<sub>2</sub></td><td>57.54 ± 7</td><td>69.67 ± 8.57</td></tr><tr><td>S<sub>1</sub> + S<sub>3</sub></td><td>55.43 ± 7.14</td><td>67.80 ± 8.74</td></tr><tr><td>S<sub>2</sub> + S<sub>3</sub></td><td>54.24 ± 7.27</td><td>66.84 ± 8.90</td></tr><tr><td>S<sub>1</sub> + S<sub>2</sub> + S<sub>3</sub></td><td>47.68 ± 4.57</td><td>55.60 ± 5.60</td></tr></tbody></table>

The Tunisian ore has a Cd content of 137 mg/kg of phosphate [30]. If such an ore is considered as a fertilizer, it would be adopted in European Union countries, where the maximum acceptable concentrations of phosphate vary from 40 to 90 mg/kg. It seems that a future limitation to Cd content will have a significant effect on the marketability of raw mineral fertilizers. Several countries have fixed legal limits on fertilizer Cd content [31]. Reducing the cadmium content in the phosphate ore early in the production process can be very advantageous [32]. In bioleaching, bacteria develop metal resistance mechanisms to Cd absorption, which may comprise exclusion by permeability barrier cell sequestration; enzymatic detoxification; reduction of the metal to less toxic forms; and pH changes which leads to the accumulation of toxic metal ions by two well-defined processes, biosorption and bioaccumulation [33,34]. Recent studies also showed that, in order to escape the metal stress, bacteria may express certain resistance genes that lead to the production of enzymes, allowing for survival under these extreme conditions [13].

### 4. Conclusions

The analysis of the composition of C(I) and C(II) layers proved the presence of carbonate-fluorapatite, illite, and montmorillonite in the C(II) layer, whereas it identified the existence of carbonate-fluorapatite, calcite, quartz, sanidine, clinoptilolite, and taramovite in the C(I) layer. Additionally, this analysis showed that the P₂O₅ and CaO contents were greater in the C(II) layer than in the C(I) layer and that the SiO₂, MgO, CO₂, Cd, Zn, and COrg contents were higher in the C(I) layer than in the C(II) layer. Moreover, the Cd bioleaching from phosphate rock samples using three bacterial strains showed a
decrease in cell viability and a change from moderately to strongly hydrophobic cells. The use of the consortium (S1 + S2 + S3) is the most powerful bio-tool for reducing the Cd content after bioleaching assays. This result showed that the isolated strains could be used as cheap and environmentally safe biological tools for bioleaching.

**Author Contributions:** Conceptualization, S.B.Y. and A.E.; validation, A.E.; formal analysis, B.M., I.T., S.B.Y. and A.E.; investigation, I.T. and B.M.; data curation, S.B.Y. and I.T.; writing—original draft preparation, S.B.Y., I.T. and A.E.; writing—review and editing, S.B.Y.; supervision, A.E., E.B.S. and B.L.; project administration, A.E. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

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