Isolation, Characterization and Identiﬁcation of a New Lysinibacillus fusiformis Strain ZC from Metlaoui Phosphate Laundries Wastewater: Bio-Treatment Assays

: The aim of the present study is to isolate, characterize and identify a novel strain ZC from the Metlaoui phosphate laundries wastewater (MPLW). The chemical characterization of this phosphate rich efﬂuent showed an alkaline pH and is saline, highly turbid and rich in suspended matter and total solids. The MPLW samples were loaded with potentially toxic metals, presented in decreasing order as follows: magnesium (5655 mg L − 1 ), potassium (45 mg L − 1 ), lead (1 mg L − 1 ), iron (0.7 mg L − 1 ), cadmium (0.5 mg L − 1 ), copper (0.3 mg L − 1 ) and zinc (0.1 mg L − 1 ). Due to the high COD/BOD 5 ratio, a poorly biodegradable organic load is underlining. The newly isolated strain was identiﬁed as Lysinibacillus fusiformis using 16S rDNA sequencing analysis. The viability of this new strain was tested in presence of the zinc, lead, cadmium, manganese and copper at 1, 10 and 100 mM. The L. fusiformis survival, under metallic stress, was inversely proportional to metal ion concentrations, while lead and zinc were the most toxic ones using MTT assay. Then, the newly isolated strain was characterized in terms of enzyme production, proteomic alteration and antibiotic resistance. The strain ZC revealed some modiﬁcations in the biochemical and enzymatic proﬁles by either the appearance or/and the disappearance of some activities. In addition, the increase in metal ions stress and concentrations was proportional to the adherence and to the hydrophobicity. The presence of the metal ions suggested the change of sensitivity to the resistance of this strain towards tobramycin, kanamycin, neomycin, netilmicin and cefoxitin, showing an increase in the MAR index . The strain ZC, used as a biological tool for MPLW treatment, showed a reduction in the metal ion contents. This reduction was due to accumulation and/or adsorption, showing a bioprocessing performance of the newly isolated L. fusiformis .


Introduction
The Company of Phosphates of Gafsa (CPG, Gafsa, Tunisia), the world's fifth largest phosphate producer, was producing up to 8 million metric tons (t) of phosphate a year until 2010. Social unrest in Tunisia, however, has subsequently affected production [1].

Study Site and Sampling
Metlaoui Phosphate laundries wastewaters (MPLW) sampling was carried out at the Metlaoui phosphate laundries, Gafsa Governorate, southwest (north latitude 34 • 20 , east longitude 8 • 22 ) of Tunisia. Sample was taken in plastic recipient, on March 2015, at the exit of the laundries and stored at +4 • C until analyses.

Physicochemical Characterization of Phosphate Laundries Wastewaters
The pH, electrical conductivity (EC) and turbidity were measured using STARTER 2100 pH meter (Ohaus, Parsippany, NJ, USA), Cond1970i conductivity meter (WTW, London UK) [4]. The chemical oxygen demand (COD), the biochemical oxygen demand (BOD 5 ), the total organic carbon (TOC), the total solids (TS), the total suspended solids (TSS) and the volatile suspended solids (VSS) were determined according to Ben Younes et al., 2013 [18]. Heavy metals and cations concentrations were determined by flame atomic absorption spectrometry (Analytik Jena NOVA 400, Jena, Germany) of samples digested with an acid mixture of HCl and HNO 3 .

Isolation of Bacteria from Phosphate Laundries Wastewaters
Nine strains were isolated from the MPLW and were preserved as pure cultures. The bacteria were isolated from MPLW supplemented with agar-agar.

Nucleotide Sequence Accession Number
The nucleotide sequence of the amplified region of DNA strain ZC was determined in this study and was submitted on 22 April 2016 to GenBank under the accession number ID: KX148607.

Enzymatic Profile of Strain ZC
The enzymatic profile of the isolated strain ZC was done according to the method described previously by Ben Kahla-Nakbi et al., 2009 [21]. The presence of several activities was determined following inoculation onto Trypticase soy agar (TSA) (Difco, Franklin Lakes, NJ, USA) by adding the following substrates: 0.2% (w/v) starch for amylase, 1% (w/v) skim milk for caseinase, 1% tween 80 for lipase, 5% (v/v) egg yolk for phospholipase (lecithinase), 1% (w/v) gelatin for gelatinase and 5% (v/v) sheep red blood cells for haemolysin. After incubation of 24-48 h at 37 • C, a positive reaction was seen by a clear halo around of the colony. Only for the amylase activity, the activity was revealed by flooding plates with iodine solution (0.2% (w/v) iodine in 2% (w/v) KI). When amylolytic enzymes were secreted by the bacteria, a clear zone could be seen around the colony. The undigested starch would be stained [22].

Determination of Heavy Metal Content Using Atomic Absorption Spectrometry (AAS) Technique in the Absence and in the Presence of ZC Strain
The metal processing ability of ZC bacterial strain was evaluated by measuring the changes in the metal ion amounts in the culture medium using atomic absorption spectrophotometer. After 24 h, a sample was taken from microcosms incubated at 37 • C and at 120 rpm for the metal ion amount determination [23].

Biofilm Formation Assays by L. fusiformis Phenotypic (Qualitative) Characterization of Slime-Producing Bacteria
Qualitative detection of biofilm formation by L. fusiformis was studied, in triplicate, by culturing the strains on Congo red agar (CRA) plates [24]. The newly isolated strain ZC, identified as L. fusiformis, was inoculated at the CRA surface plates (0.8 g Congo red, 36 g sucrose (Sigma-Aldrich, Buchs, Switzerland) in 1 L of brain heart infusion agar), and incubated for 24 h at 37 • C and overnight at room temperature. Slime-producing bacteria appeared as black colonies, whereas non-slime producers remained unpigmented [25].

Characterization of Semi-Quantitative Slime-Producing Bacteria
Biofilm production by L. fusiformis was determined using a semi-quantitative adherence assay on 96-well tissue culture plates [26]. Strain was grown in Trypticase soy broth supplemented with various concentrations (1, 10 and 100 mM) of metal ions at 37 • C. The optical density (OD 600 ) of the bacterial culture was measured at 600 nm. Two hundred µL of cell suspensions (OD = 0.3) was transferred in a U-bottomed 96-well microtiter plate (Nunc, Roskilde, Denmark). Each strain was tested in triplicate. Tryptic soy broth (TSB) (Difco, Franklin Lakes, NJ, USA) was used as control. After incubation at 37 • C for 24 h, the cultures were removed and the wells were washed twice with phosphate-buffered saline (PBS) (7 mM Na 2 HPO 4 , 3 mM NaH 2 PO 4 and 130 mM NaCl at pH 7.4) to remove non-adherent cells and dried in an inverted position. Adherent bacteria were fixed with 95% ethanol and stained with crystal violet (1%) (Merck, France) for 5 min. The excess stain was rinsed, and the wells were washed three times with sterile distilled water. Then, the water was cleared, and the microplates were air-dried. The optical density of each well was measured at 570 nm (OD 570 ) using an automated Multiskan reader (Gio. de Vita E C, Rome, Italy). Biofilm formation was interpreted as highly positive (OD 570 ≥ 1), low-grade positive (0.1 ≤ OD 570 < 1) or negative (OD 570 < 0.1) [27]. Quantitative adherence assays were performed with human oral cavity epidermoid carcinoma (KB) cell line [28]. KB cells (2 × 10 5 ) were seeded and grown overnight in Minimal Essential Medium (MEM) with Earle's salts and 10% fetal bovine serum in 96-well microtiter plates at 37 • C with 5% CO 2 . L. fusiformis strain was grown overnight in TSB supplemented with metal ion solutions (1, 10, 100 mM) at 37 • C and 150 rpm. The human cells monolayers were inoculated with L. fusiformis strain (10 8 UFC/mL) and incubated at 37 • C in 5% CO 2 for 60 min. Then, bacterial suspension was removed to exclude the unattached bacteria. The monolayers of KB cells were washed 3 times with Dulbecco's Modified Eagle Medium (DMEM), and 1 mL Triton X-100 in PBS was added for 5 min at room temperature to release the bacteria from the cells. The number of bacteria was estimated by plating of the serial dilutions of bacterial suspension on PBS and counted in nutrient agar plate.
2.6.6. Cell Surface Hydrophobicity Hydrophobicity was measured by the hexane, decane and chloroform partitioning method [29]. Bacterial cells, grown overnight in TSB supplemented with different concentration of metal ions (1, 10 and 100 mM), were washed with PBS and re-suspended in 4 mL of PBS, and the absorbance (OD 540 ) was determined. One ml of hexane, decane and chloroform were added to each cell suspension for 10 min and was re-incubated at 37 • C for 30 min. The aqueous layer was removed and aerated to remove all traces of hexane, decane and chloroform, and absorbance (OD 540 ) was measured using UV spectrophotometer (UV-1800 Shimadzu, Kyoto, Japan) against a hexane-, decane-and chloroform-extracted PBS blank. The hydrophobicity index was expressed as the ratio of absorbance of the hexane-, decane-and chloroform-extracted sample to absorbance of the sample before extraction.

Antibiotic Susceptibility Testing
The Kirby-Bauer disk diffusion assay was used to test resistance of L. fusiformis against seven antibiotics (penicillin (6 µg), kanamicyn (30 µg), neomycin (30 µg), netilimicin (30 µg), tetracyclin (30 µg), tobramycin (10 µg), cefoxitin (30 µg)), in the absence and presence of metallic ions using standardized potencies [30]. The selected antibiotics were based on two criteria, the antibiotics action mechanism (β-lactamas, protein synthesis inhibition, DNA replication inhibition and disruption of the folic acid pathway) as well as the most common antibiotic resistance pattern observed. Multidrug resistance (MDR) is defined as non-susceptibility to at least three or more antimicrobial agents. The multiple antibiotic resistant (MAR) index for each isolate was calculated as a ratio between the number of antibiotics to which the isolate is resistant to the total number of antibiotics against which the isolate was tested [30].

Bio-Treatment Assays of Phosphate Laundries Wastewaters
The phosphate laundries wastewater bio-treatment assays were carried out in 500 mL Erlenmeyer flasks. First, 250 mL of autoclaved MPLW were inoculated with a pre-culture of L. fusiformis in LB medium. The cultures were incubated in a rotary incubator for one week at 30 • C and 150 rpm. The potential bio-treatment of the newly identified strain was assessed by analyzing the amount of potentially toxic metal ion concentrations in the bio-treated phosphate laundries wastewater. The removal efficiency was measured as follows: Removal efficiency (R eff (%)) = [(Ci−Cf) × 100]/Ci (1) where Ci and Cf are the concentration of initial and final metal ions in the solution (mg L −1 ), respectively.

Statistical Analysis
Statistical analysis was performed using the S.P.S.S. 13.0 statistics package for Windows (Redmond, WA, USA). The differences in the degree of adhesion assay were examined by the Friedman test, followed by the Wilcoxon signed ranks test. p values of < 0.05 were considered as significant. All experiments were performed in triplicate.

Physicochemical Characteristics of Phosphates Laundries Wastewater
The first part of the experimental survey involved the characterization of Metlaoui phosphates laundries wastewater (MPLW) in terms of the conventional parameters. The physicochemical characteristics of the MPLW are summarized in Table 1. A considerable variability in the MPLW characteristics, released by phosphate laundries in Gafsa, Tunisia, and in the world, has been noticed [1]. The studied MPLW presents relatively alkaline pH (pH = 7.5) and slightly saline (EC = 3.5 mS cm −1 ), which favors the development of microorganism purifiers in aerobic and anaerobic environments [31]. This salinity was associated with the MPLW richness in chlorosity (1.1 g L −1 ). This effluent contained high amounts of total suspended solids (TSS = 61.8 g L −1 ), resulting in an elevated value of their turbidity (503 NTU). The values of the total suspended solids obtained were high, which revealed that there were suspended particles in the water samples analysed that invariably decreased the transparency and showed that the samples were highly polluted [31]. In addition, the total organic carbon (TOC), an important parameter in the assessment of organic pollution of water, was high (TOC = 3.7 g L −1 ) and exceeded the ranges fixed by Tunisian and international standards (Table 1) [32,33]. Biodegradability is the capacity of microorganisms to degrade organic matter. In addition, the COD value reached 1155 mg L −1 , and the BOD 5 was 280 mg L −1 . Hence, the value of the MPLW COD/BOD 5 ratio is superior to the border three, underlining an organic load that is poorly biodegradable. The reported DBO 5 /DCO provides important indications on the origin of the pollution of the employed waters and the proper treatment to be accomplished [31]. It was noticed that the COD, BOD 5 and TSS were all exceeding the previously reported ranges for industrial wastewaters [6]. Similar results were reported by many researchers [4,6]. The TSS/DBO 5 ratio explains that the material in suspension unsettles the oxygenation and bacterial activity. This report is weak (0.2) at the MPLW effluent.
Moreover, the MPLW effluent seems to be highly loaded with metal ions. In this study, heavy metal ion concentrations were not high, being attributed to the dilution effect when using large amounts of water. In fact, the average amounts of Mg (5655 mg L −1 ), Pb (1 mg L −1 ) and Cd (0.5 mg L −1 ) are considerably higher than the required limits [32,33]. However, the K (45 mg L −1 ), Fe (0.7 mg L −1 ), Cu (0.3 mg L −1 ) and Zn (0.1 mg L −1 ) contents were relatively comparable to the limit fixed by international standards for wastewater discharges (Table 1). Metal ion concentrations in phosphate laundries wastewaters depend on the water quantity used that generates the waste. In fact, effluents may contain various chemical pollutants that contribute to high-suspended solids contents, high COD and BOD concentrations and intensive color as well as other soluble substances. This process produces multi-component wastewaters, which usually cause difficulties and inhibitory behaviours in treatment processes [18]. In addition, the Metlaoui phosphate laundries release a fine fraction (<70 µm) in effluents directly into the hydrographic network. Such effluents were rich in organic matter, which promotes an additional capacity to trap toxic elements [4,6]. It has been reported that these effluents contain high concentrations of sulfates, fluorine and various metals [36]. In addition, many negative effects of mud on the structure and permeability of soils bordering laundries were reported [6]. On the other hand, it was reported that the MPLW effluents of the laundries of Metlaoui, Gafsa, contain various metal contaminants, such as cadmium, chromium, copper, nickel and strontium [5]. The accumulation of toxic metal ions in the soils receiving phosphate industries effluents as well as their phytotoxicity were equally demonstrated [4,5]. In fact, exposure to many elements present in the MPLW effluents was associated with several health repercussions, such as affecting the human respiratory system and causing dental fluorosis due to fluoride, while mining activities might lead to cancer [4,6].

Phylogenetic Identification of Strain ZC
Nine bacterial strains were screened from the MPLW effluent. Among these isolates, strain ZC was selected for its high minimal inhibition concentrations towards Zn 2+ , Cu 2+ , Pb 2+ , Mg 2+ and Cd 2+ metal ions and its overproduction of exopolysaccharides in stressed conditions (data not shown). In order to identify the ZC isolate, purified DNA was amplified by the polymerase chain reaction using the universal bacterial Bac27F and Univ1492R primers [19]. The amplified DNA nucleotide sequence was about 1223 bp, and it was subjected to BLAST analysis. The DNA was extracted using an extraction kit "Wizard Genomic DNA Purification Kit" (Promega, Madison, WI, USA). The sequences used in the phylogenetic analysis were based on the fragments of sequences of the 16S rDNA of the type strains obtained from the ribosomal data project (RDP) and GenBank databases. Pairwise evolutionary distances were calculated using the method of Jukes and Cantor [37]. A dendrogram was constructed using the neighbor-joining method with MEGA-X software (State College, PA, USA) [38] (Figure 1). Confidence in the topology of the dendrogram was determined using 100-bootstrapped trees. nomic DNA Purification Kit" (Promega, Madison, WI, USA). The sequences used in the phylogenetic analysis were based on the fragments of sequences of the 16S rDNA of the type strains obtained from the ribosomal data project (RDP) and GenBank databases. Pairwise evolutionary distances were calculated using the method of Jukes and Cantor [37]. A dendrogram was constructed using the neighbor-joining method with MEGA-X software (State College, PA, USA) [38] (Figure 1). Confidence in the topology of the dendrogram was determined using 100-bootstrapped trees.

MTT Assay
The tolerance of the L. fusiformis strain to metal ions was examined. The results showed a decrease in cell viability with an increase in metal ion concentrations (Figure 2). The newly isolated strain, identified as L. fusiformis, was able to survive in the presence of metal ions containing media at a concentration equal to 1 mM as the control. In fact, increasing the metal ion concentrations, the viability was different from the control and decreased drastically after 24 h of incubation. In fact, the viability of the newly isolated bacteria with different metal ion concentrations within 72 h in TSB broth microcosms showed its viability (not exceeding 30%) in spite of the decrease in cell number after 72 h of incubation. In addition, a drop in the number of bacteria was noticed from the first day of incubation and for all metal ion presences and concentrations. The decrease was significant at 100 mM and reached about 10-30% for all metal ions. This decrease was also proportional to the incubation time. The presence of metal ions was toxic to the bacteria in the medium. The toxicity effect of the metal ions was classified in a decreasing order as follows: Pb, Zn > Cd > Cu > Mg.

MTT Assay
The tolerance of the L. fusiformis strain to metal ions was examined. The results showed a decrease in cell viability with an increase in metal ion concentrations (Figure 2). The newly isolated strain, identified as L. fusiformis, was able to survive in the presence of metal ions containing media at a concentration equal to 1 mM as the control. In fact, increasing the metal ion concentrations, the viability was different from the control and decreased drastically after 24 h of incubation. In fact, the viability of the newly isolated bacteria with different metal ion concentrations within 72 h in TSB broth microcosms showed its viability (not exceeding 30%) in spite of the decrease in cell number after 72 h of incubation. In addition, a drop in the number of bacteria was noticed from the first day of incubation and for all metal ion presences and concentrations. The decrease was significant at 100 mM and reached about 10-30% for all metal ions. This decrease was also proportional to the incubation time. The presence of metal ions was toxic to the bacteria in the medium. The toxicity effect of the metal ions was classified in a decreasing order as follows: Pb, Zn > Cd > Cu > Mg.
Mine wastewater can profoundly influence biological systems. For example, species diversity and the total biomass composition in aquatic and terrestrial ecosystems can be affected by heavy metal contamination [39]. The newly isolated L. fusiformis from MPLW is able to adapt and survive under extremely stressful conditions.

Determination of Heavy Metal Content Using Atomic Absorption Spectrometry (AAS) Technique
The determination of metal ion contents, using AAS, was assayed to show whether the bacteria, in each microcosm accumulate and/or adsorb these toxic ions. By analyzing the results illustrated in Table 2, it is clear that strain ZC can accumulate and/or adsorb a small content of all the studied metal ions. In fact, the percentage of accumulation and/or adsorption did not exceed 20% for all metal ions at a concentration of 1 mM with the most minor ones for cadmium (4.6%). Increasing the concentration at 10 mM, the accumulation percentage was proportional to the concentration. It ranged between 11 and 35%. Similar results were demonstrated at 100 mM. The stressed strain ZC accumulated a high amount of metal ions exceeding 45%. In fact, it has been found that there is a decrease in the level of trace metals in the culture media, regardless of the concentration. The ZC strain manifested a bioaccumulation performance according to the following increasing order of metal ions: Cu > Zn > Cd > Mg > Pb. This accumulation into ZC strain was confirmed by the decreasing amount of metal ions in the supernatant before sonication.  Biosorption is defined as an interaction between the living and non-living microorganisms and the metallic ions in the system. This process is used for removing metal or metalloid species, compounds and particulates from solutions by biological materials [12,40]. The most important types of biosorbents that have been developed from diverse raw biomass are bacteria due to their ubiquity, size, ability to grow under controlled conditions and resilience to environmental conditions (e.g., Pseudomonas fluorescens; Bacillus safensis; Pseudomonas aeruginosa) [41,42]. L. fusiformis is able to adapt and survive under extremely stressful conditions. In addition, we find that the number of L. fusiformis cells decrease with the increase in metal concentration, but they remain viable. Thus, this resistance could be explained by the metallic pollution induced by the effluents of the Company of Phosphate Gafsa (CPG). Previous studies have noted that the high frequencies of metal-resistant germs encountered at a given site are related to the selective pressures exerted by heavy metals within this site [32,43]. Similarly, contact with other bacterial organisms at such frequency due to the process of transfer of genes enabling resistance to heavy metals, which are mainly located on plasmids between strains [33]. Generally, to survive under metal stress conditions, bacteria have adopted various types of heavy metal resistance mechanisms, such as alteration of their membrane by which bacteria protect their essential cellular components sensitive to metals. Some bacteria have a membrane or envelope that is capable of passively adsorbing high levels of dissolved metals, generally via a charge-mediated attraction [44,45], accumulation and complexation in the metal ion inside the cell, reduction of the toxicity of this metal [16], enzymatic transformation [46], as well as the decrease in the level of metal ions in the culture media, whatever the concentration. This confirms that there is intracellular or extracellular sequestration. In fact, many bacteria have developed a cytosolic sequestration mechanism for protection from metals through converting heavy metals into more inoffensive forms using cytoplasmic proteins, such as metallothioneins, to bind, sequester or store metals [16,47,48]. In the bacterium, a fine regulation for the maintenance (homoeostasis) and the control of their intracellular concentration [46] prevents the expulsion of essential metals present at homeostatic concentrations or the entry of metals in toxic amounts. Furthermore, extracellular polymers and siderophores produced by bacteria to withstand heavy metals can trap or precipitate metal ions in the extracellular environment [49,50]. These compounds bind heavy metals and subsequently detoxify metals simply via complex formation or by forming an effective barrier surrounding the cell [51]. Metals might also attach to, or precipitate at, bacterial cell surfaces by interactions involving proteins or cell-associated polysaccharides [32].

Enzymatic Changes
The newly isolated ZC strain identified as L. fusiformis was tested to produce exoenzymes such as amylase, lipase, gelatinase, caseinase, lecithinase and haemolysin using specific media on agar plates ( Table 3). The control strain could produce gelatinase, caseinase, amylase and lecithinase and was unable to secrete lipase and haemolysin. The production was revealed by the appearance of a halo around the colony in each specific medium. Under the metallic stress, the new strain produced a new enzymatic activity named lipase. On the other hand, the loss of caseinase activity in the presence of 100 mM of copper and magnesium has been noticed, as well as lecithinase and gelatinase at 10 and 100 mM of zinc, respectively. In fact, gelatinase and lecithinase, zinc-metalloproteinases, are inhibited at high concentrations of zinc [52,53]. The mechanism of enzyme inactivation by metals is not completely understood. It is assumed that metal ions bind to specific sites, causing conformational changes that inactivate the catalytic function of enzymes. The non-competitive inhibition by other heavy metal ions is attributed to the binding of the ion to a site distinct from the active site [54]. Consequently, zinc is essential for the activity of these enzymes. It can either be directly active at their catalytic site or intervene in their conformation.
The biochemical modifications, by the appearance of new enzymatic activities, observed in the stressed L. fusiformis were a form of adaptation to the stressful conditions of the environment. This new enzymatic activity is the result of the expression of genes involved in survival under the stressful conditions of the environment. The high concentration of zinc can also be at the origin of the loss of some characters by the repression of the genes, which code for this enzyme. Transcriptional regulation is under the control of many proteins, which bind to the promoter sequences of genes, thus preventing their production of the transcripts. When the genes are exposed to metals, they bind these metals, inducing an allosteric change so that RNA polymerase can transcribe the target genes [45].

Qualitative Slime Production
The qualitative detection of biofilm formation was studied by culturing L. fusiformis on congo red agar. The newly isolated strain ZC shows colonies with a red center and a lighter outer area (Figure 3). These later properties showed its negative slime character. After the metal ions effect, the ZC strain changes its phenotypic profile and becomes brown. Indeed, these changes are considered to be a variable phenotype [55]. Slimeproducing bacteria appeared as black colonies, whereas non-slime producers remained unpigmented. This difference between the strain in the normal and stressed state is a state of transition to biofilm formation. It could be proposed that the bacterium is developing a variety of resistance mechanisms to neutralize the toxic effect of the metal. In fact, biofilmcombining microorganisms have been shown to play a crucial role in the nutrient cycling and biodegradation of environmental pollutants [32,56]. In addition, biofilm bacteria are generally embedded in a polymeric extracellular substance. However, this substance, called slime, seems to play an important role [32,56]. In addition, exopolysaccharide can also act as an ion exchange and is able to positively sequester the charged pollutants [57].

Qualitative Slime Production
The qualitative detection of biofilm formation was studied by culturing L. fusiformis on congo red agar. The newly isolated strain ZC shows colonies with a red center and a lighter outer area (Figure 3). These later properties showed its negative slime character. After the metal ions effect, the ZC strain changes its phenotypic profile and becomes brown. Indeed, these changes are considered to be a variable phenotype [55]. Slime-producing bacteria appeared as black colonies, whereas non-slime producers remained unpigmented. This difference between the strain in the normal and stressed state is a state of transition to biofilm formation. It could be proposed that the bacterium is developing a variety of resistance mechanisms to neutralize the toxic effect of the metal. In fact, biofilm-combining microorganisms have been shown to play a crucial role in the nutrient cycling and biodegradation of environmental pollutants [32,56]. In addition, biofilm bacteria are generally embedded in a polymeric extracellular substance. However, this substance, called slime, seems to play an important role [32,56]. In addition, exopolysaccharide can also act as an ion exchange and is able to positively sequester the charged pollutants [57].

Quantitative Adhesion and Culture Cells Adherence Assays
Quantitative adherence assays were performed with a human oral cavity epidermoid carcinoma (KB) cell line. The strain is considered weakly biofilm positive (0.1 < OD570 < 1) in the absence of any stress and in the presence of various metal ions such as Cu, Cd, Mg, Pb and Zn. In fact, the unchangeable property was improved by a significant (p < 0.05) increase in adherence in presence and with an increase in metal ions stress and concentrations, respectively (Table 4). In addition, the obtained results showed that the stress applied leads to a significant (p < 0.05) increase in adhesion (Table 4). In fact, the unstressed A B Figure 3. Induction of slime production by metallic stress: Slime production was analysed using congo red agar plates. Lysinibacillus fusiformis ZC strain was cultured (A) with and (B) without metal ions (1, 10 and 100 mM) on congo red agar plates for 24 h at 37 • C. Four independent experiments were conducted, and one set of representative results is shown.

Quantitative Adhesion and Culture Cells Adherence Assays
Quantitative adherence assays were performed with a human oral cavity epidermoid carcinoma (KB) cell line. The strain is considered weakly biofilm positive (0.1 < OD 570 < 1) in the absence of any stress and in the presence of various metal ions such as Cu, Cd, Mg, Pb and Zn. In fact, the unchangeable property was improved by a significant (p < 0.05) increase in adherence in presence and with an increase in metal ions stress and concentrations, respectively (Table 4). In addition, the obtained results showed that the stress applied leads to a significant (p < 0.05) increase in adhesion (Table 4). In fact, the unstressed strain was weakly adherent, and in the presence in metal ions stress, the adherence became strong. In fact, bacteria have a natural tendency to adhere to surfaces as a survival mechanism under adverse conditions. Bacterial colonization of solid surfaces has been described as a basic ploy in a wide variety of environments [57]. Generally, biofilms, composed of extracellular polymeric substances, have also been reported to adsorb heavy metals [32,50].

Cell Surface Hydrophobicity
Hydrophobicity was measured by the hexane, decane and chloroform partitioning method. The obtained results (Table 5) indicate that strain L. fusiformis is weakly hydrophobic in the absence of metal ions. A significant variation (p < 0.05) in the hydrophobicity of L. fusiformis depends on the type and the concentration of metal ions and also of the used solvent. Indeed, the hydrophobic state was changed from the weakly to the hydrophilic state using chloroform as the solvent. On the other hand, the hydrophobicity increased when decane was used. In addition, an increase in hydrophobicity was noticed using hexane in the presence of Zn and Mg, but there was also a decrease in the presence of Cu. For microorganisms, hydrophobicity is considered as a virulence factor that detracts from the adhesion of microorganisms to biotic and abiotic surfaces. This variation in hydrophobicity is probably due to the metal stress effect as well as the nature of the solvent. Indeed, the surface properties of stressed strains are different from those of logarithmically growing strains. Among these properties, hydrophobicity is the most important one. This change is expected to increase the chances of survival in adverse conditions. Under adverse conditions, the bacterium regulates its protein synthesis by reorganizing the membrane, degrading certain proteins and synthesizing new "stress" proteins [58]. Generally, the modification of properties of the cell surface of bacteria plays a major role in resistance to harsh conditions as well as in removal of contaminants [59,60].

Antibiotic Sensitivity of Bacteria
The sensitivity or antibiotic resistance of L. fusiformis mentioned in Table 6 varied according to the metal ion as well as its concentration. The ZC strain was tested against seven antibiotics. Two were β-lactams (penicillin and cefoxitin), three inhibited protein synthesis (kanamycin, tetracycline and netilmicin) and two inhibited DNA replication (neomycin and tobramycin). The new ZC strain was sensitive to the majority of the used antibiotics except for penicillin. In fact, the incubation of the strain with metal ions in the different microcosms, revealed the change of sensitivity to the resistance of this strain towards tobramycin, kanamycin, neomycin, netilmicin, and cefoxitin. MAR index is defined as resistance to ≥3 antibiotics; in this study, seven antibiotics were tested. Consequently, a MAR index of 0.7 and 0.8 translate to antibiotic resistance towards five and six antibiotics respectively. The MAR index of the ZC strain fell into this range within incubation with metal ions at different concentrations (Table 6). Only, in presence of 1 mM of manganese, ZC strain was resistant to all the seven tested antibiotics, showing its multi-resistance. This L. fusiformis resistance can be essentially enabled by four mechanisms: the reduction of the membrane permeability, the efflux, the modification of the target and the inactivation of the antibiotic. As a result, metal contamination functions should be taken seriously as a selective agent for the proliferation of antibiotic resistance. Indeed, previous research has shown mechanisms including co-resistance to heavy metal ions and antibiotics [61]. The genetic determinants involved in these mechanisms are present in the bacterial genome [62]. Furthermore, different studies show a positive correlation between the presence of metal ions in an ecosystem and the resistance to both metal ions and antibiotics in native bacteria, regardless of the metal or the antibiotic [30,63].

Bio-Treatment Assays of Phosphate Laundries Wastewater Using Lysinibacillus fusiformis
The richness of MPLW in cultivable mesophilic microflora (38 × 10 4 CFU mL −1 ), used as a parameter, encourages the bio-treatment processing assay of the effluent using an indigenous strain. After successive sub-cultures on autoclaved MPLW supplemented with agar-agar, the ZC isolated strain was used in order to study their bioprocessing performance. The variations of the used metal ions concentration in phosphate laundries wastewater induced by reacting with new ZC isolated strain are presented in Table 7. The result showed a very high decrease in the concentration of Cu, Cd, Pb, Zn and Mg after adding L. fusiformis. The results showed different performance rates of bioprocessing with the addition of the metal ions. Indeed, results showed that the removal efficiency (R eff ) of magnesium (Mg) was the mostly reduced element followed by lead (Pb) and zinc (Zn) with a R eff equal to 86-87%, then cadmium (Cd) and finally copper (Cu) with R eff equal to 82.3% and 80.6%, respectively. The bio-treatment assays of phosphate laundries wastewater by L. fusiformis new isolate showed a very important decrease in the concentration of the heavy metal ions. Indeed, biosorption is becoming a potential alternative to the existing technologies for the removal and/or recovery of toxic metals and it has been reported that some bacterial strains naturally have ability to uptake heavy metal ions, such as Cu, Zn and Mg from solutions at a relatively lower concentration. The bioremediation process is an approach for removing these toxic elements from the polluted sites and/or transforming into less toxic form by applying live or dead microbes. Microorganisms are omnipresent in the environment and play a pivotal role in the biogeochemical cycles [64]. The bioremediation technique can be implemented in in situ and ex situ methods. The remediation is carried out in the site of contamination by stimulating the growth of indigenous microbes, supplying adequate nutrition or application of engineered microbes [65]. Therefore, many organisms have been used from different raw biomass, such as bacteria (e.g., Pseudomonas fluorescens; Bacillus safensis; Pseudomonas aeruginosa; Bacillus thuringiensis) [41,42,66], fungi (e.g., Botrytis cinerae) [12,67] and algae (e.g., Anabaena sphaerica) [68]. However, there are several advantages of microbialbased biosorption in the removal of metal ions, such as the high metal removal efficiency thanks to their selectivity towards particular metal [64].

Conclusions
In this study, a new strain ZC was isolated from the Metlaoui phosphate laundries wastewater (MPLW) and identified as L. fusiformis. Indeed, the MPLW was especially rich in various heavy metal ions and had a high COD/BOD 5 ratio underlining a poorly biodegradable organic load. The newly isolated strain showed a decreasing viability and an increasing adherence in presence of metal ions at 100 mM. In addition, the enzymatic and biochemical profiles as well as resistance or sensibility to antibiotics was changed. The strain ZC, used as a biological tool for MPLW treatment, showed a reduction of the metal ion contents. This reduction was due to accumulation and/or adsorption showing a bioprocessing performance of the newly isolated L. fusiformis. This potential can be used safely for the bio-treatment of effluents.