1.                   Introduction

Textile Wastewaters (TWWs) are considered as one of the major sources of pollution due to the great demand for textile products resulting proportional increase in production and application of synthetic dyes. Azo dyes represent the most common group of chemical dyes constituting 60-70% of more than 10,000 dyes used in textile industries [1]. It is estimated that about 2% and 10–15% of azo dyes are lost during manufacture and dyeing processes, respectively [2]. Release of theses dyes into the environment causes an adverse impact on different ecosystems because that they are considered to be toxic to aquatic biota and are reported to be carcinogenic to humans [1]. Although their contribution to organic load can sometimes be insignificant, even the presence of low levels of synthetic dyes would impart an intense colour to the TWWs [1,3]. Therefore, effective treatment of dye-containing wastewaters before discharge into the environment should be emphasized.

Compared with physical and chemical methods, biological treatment processes are generally considered as better alternatives for treatment of azo dye-containing wastewaters due to their lower cost, higher efficiency and less secondary pollution [4]. Generally, the TWW generated in reactive dyeing process not only marked by high colour, and pH but also carry a high load of salts. Although the majority of microorganisms were sensitive to harsh industrial conditions, some bacterial extremotolerant species still had shown the ability to degrade synthetic dyes or raw TWWs under alkaline and saline conditions [1,5,6]. The isolation and selection of highly efficient extremophilic bacteria are among the most important factors for effective biological treatment of TWWs [7-10]. Response Surface Methodology (RSM) was proven to be a powerful tool to optimize biodecolourization processes [5,11-14].

In this study, an alkali-halo tolerant bacterial strain capable of producing laccase and decolourizing various textile azo dyes was isolated, identified and characterized. Bezactiv blue S-Matrix 150 was chosen as the model azodye for further investigating the effects of different parameters on dye decolourization by means of RSM.

2.                   Materials and Methods

2.1.              Dyes, Media and Chemicals

Bezactiv Blue S-Matrix 150 and Tubantin brown GGL used in this study were supplied from a textile factory in Nabeul, Tunisia and was of commercial quality. Stock dye solution was prepared at concentration of 1000 mg/L (w/v) sterilized by filter and added to the media in all manipulations. λ max of the dyes were determined in diluted dye aqueous solutions by using a scanning UV-vis spectrophotometer. Tryptic Soy Broth (TSB) (Sigma, 22092) and Tryptic Soy Agar (TSA) (Sigma, 22091) were used in the experiments. All chemicals used were of the highest purity available and of analytical grade.

2.2.              Sampling, Isolation and Screening of Laccase Producing Extremophilic Bacteria

A composite soil sample from Ksar Ghilane oasis (N 32°59′012′′, E 09^°38′072′′), an arid system in Tunisian desert, was prepared aseptically from five subsamples (1-10 cm deep) and collected from the arms and center of an X (each arm was 1 m in length). One cm soil from the ground surface was firstly removed to avoid contamination during sampling procedure. Samples were then transported to the laboratory in a cool box and stored at 4^°C prior to processing [15].

Enrichment technique was applied for the isolation of moderately haloalkaliphilic bacteria as reported by El-Hidri et al. [16]. Plate assay method was performed to confirm the presence of laccase [5]. Enzyme detection was performed using TSA medium amended with 0.2% of 2,6 dimethoxyphenol (DMP) as enzyme substrate. The presence of brick colour around the colonies was considered as DMP oxidizing laccase secreting organism. Dye decolourization activities was revealed on TSA and TSB containing 100 mg/L of the dyes [17].

2.3.              Enzyme Analysis

The laccase activity was measured by monitoring the oxidation of 5 mM DMP buffered with 50 mM phosphate (pH 8.0) at 469 nm for 1 min. To calculate enzyme activity an absorption coefficient of 27,500 M^-1cm^-1 was used. One unit of laccase activity was defined as the amount of enzyme required to oxidize 1 µM of 2,6-DMP oxidized per minute [5].

2.4.              Molecular Identification and Phylogenetic Analysis of Selected Bacterium

The 16S rRNA gene from pure cultures was amplified using the following universal primers: S-D-Bact-0008-a-S-20/S-D-Bact-1495-a-A-20 according to the procedure described previously by Cherif et al. [18]. The 16S rRNA gene sequencing has been carried with an automated capillary ABI Biosystem 3130. Obtained sequences were initially compared to those available in GenBank database using BLAST (http://www.ncbi.nlm.nih.gov). Phylogenetic dendrograms were constructed by the neighbor-joining method and trees topology was evaluated by performing bootstrap analysis of 1000 data sets using MEGA 6.06 (Molecular Evolutionary Genetics Analysis) [5]. 

2.5.              Decolourization Experiments and Optimization Study

1 ml of 1.0 % v/v (O.D. 600 nm ≈ 0.9) inoculum from the mother liquid culture was used to inoculate 50 ml of TSB medium contained dye and then flasks were incubated at 37^°C on a rotatory shaker (150 rpm). The culture was centrifuged at 10000 rpm to separate the bacterial cell mass. The decolourization was quantified by measuring the decrease in absorbance of the dye using UV-Vis Spectrophotometer. Dyes of Bezactiv Blue S-Matrix 150 and Tubantin brown GGL had λmax values of 605 and 430 nm, respectively and then dye decolourization (%) was calculated.

A CCD consisting of 30 experiments was chosen for the optimization of Bezactiv Blue decolourization. Four independent variables, namely dye concentration (X₁), pH (X₂), NaCl (X₃) and incubation time (X₄), were evaluated at three levels (Table 1), and the percentage of Bezactiv Blue decolourization was the dependent variable (response). The following equation was used to establish the quadratic model:

Y = b₀ + b₁ X₁ + b₂ X₂ + b₃ X₃ + b₄ X₄ + b₁₁ X₁²+ b₂₂ X₂²+ b₃₃ X₃² + b₄₄ X₄² + b₁₂ X₁ X₂ + b₁₃ X₁X₃ + b₂₃ X₂ X₃ + b₁₄ X₁X₄ + b₂₄ X₂ X₄ + b₃₄ X₃ X₄

where Y is the response (% decolourization); Xi and Xj are uncoded independent variables; and b₀, b_j, b_jj and b_jk are intercept, linear, quadratic and interaction constant coefficients, respectively.

The FTIR analysis was done on Perkin Elmer, spectrum one instrument in the mid IR region of 400– 4,000 cm^-1 with a scan speed of 16 (Spectrum One, Perkin Elmer, USA) as described by Si et al. [19].

2.6.              Statistical Analysis

The generation and the data treatment of the CCD were performed using NemrodW software [20].

3.                   Results and Discussion

In the present investigation, 53 alkali-halotolerant bacteria (3% NaCl, pH 8), isolated from Ksar Ghilane oasis were screened for laccase activity using the chromogenic screening method on DMP supplemented TSA medium [5]. Of the 53 isolates, 16 formed brown zone around and above the colony which is a positive confirmation for their laccase activity secretion. Laccase producing isolates were subjected to secondary screening to investigate their ability to decolourize two synthetic dyes namely, Bezactiv Blue S-Matrix 150 and Tubantin brown GGL. The isolates G7, G9, G12, G18, G19, G21, G22, G25, G27, G36, G45, G48, G49 were not able to decolourize both dyes (Table 1). In contrast, the highly laccase-producing isolate G2 was able to completely decolourize both tested dyes. Bezactiv Blue S-Matrix 150 was decolourized more rapidly than Tubantin brown GGL. Quantitative experiments in shake flask culture showed a laccase activity of 0.662±0.029 U/mL with complete Bezactiv Blue decolourization on 48h. The decolourization ability of laccase producing G2 against the two textile dyes are shown in (Figures1a-c).

The 16S rRNA gene of the isolate G2, was amplified, sequenced and submitted to GenBank. The obtained sequence with accession number MG760722 was compared with those in the National Center for Biotechnology Information Nucleotide Sequence database by using the BLAST algorithm. Based on the phylogenetic analysis G2 strain was identified as Pseudomonas resinovorans via partial sequencing of 16s rRNA Figure 1d. Other pseudomonas strains were also reported for their ability to decolourize textile dyes in particular, Pseudomonas aeruginosa [7,21,22], Pseudomonas fluorescens) [23] and Pseudomonas extremorientalis [5].

A CCD was chosen to determine the optimum requirement of enzyme (X₁), dye (X₂), salt (X₃) and time (X₄) for maximum dye decolourization by selected strain G2 (Table 2).

The mathematical expression of the relationship to the decolourization of Bezactiv blue S - Matrix 150 (Y) with the variables X₁, X₂, X₃ and X₄ is as follows:

Y = 46.86 – 13.05 X₁ – 17.37 X₃ – 7.73 X₄ - 25.35 X₁² + 16.00 X₂² + 16.85 X₃² + 6.57 X₁X₂ - 14.14 X₁ X₃ – 3.87 X₂X₄ + 6.34 X₃ X₄

Where Y are the G2 decolourization response (%); Xj: system variables (correspond to the different factors influencing the decolourization of bezactiv blue) and b₀, b_j, b_jk and b_jj: significant model coefficients.

ANOVA of the regression model demonstrated high significance of the model and insignificant lack of fit value (Table 3). The linear factors of X₁, X₃ and X₄; quadratic factor of X₁, X₂ and X₃ and interaction terms X₁₂, X₁₃, X₂₄, and X₃₄ were found to be significant indicating that the model terms are limiting factors for BEZACTIV blue S-Matrix 150 decolourization (Table 4). The regression equation obtained indicated the R-Squared, Adjusted R-Squared and Predicted R-Squared values of 0.989, 0.977 and 0.941 respectively, suggesting an adequate adjustment of the quadratic model to the experimental data and indicating that the model could explain 98.9% of the variability in the response.

The contour plots and response surface curves for the predicted response Y (Bezactiv blue decolourization yield), based on the second-order model were shown in Figure 2. They provided useful information about interactions between dye concentration, pH, salinity and incubation time and allowed an easy interpretation of the CCD results and prediction of the optimal levels of each variable for maximum Bezactiv blue decolourization. Indeed, Bezactiv blue decolourization enhanced by increasing dye concentration up to middle level (150 mg/L) and decreasing the NaCl concentration and incubation time. The decreasing dye decolourization at higher levels was probably as a result of possible enzyme inactivation at such high dye concentrations [5]. Decolourization of Bezactiv blue dye by G2 seems to be active at alkaline pHs (8-10). This result is advantageous to biologically treat TWWs because one of the most important characteristic of TWWs is their alkalinity [24].

The optimum decolourization conditions of BEZACTIV blue S-Matrix 150, carried out numerically by using NemrodW software, are dye concentration 150 mg/L, pH 10, salt concentration 1% and incubation time 24h. The expected value of BEZACTIV blue decolourization yield was y_op= 97.5% ± 2.5. Additional experiments were carried out under the selected optimal decolourization conditions. It led to BEZACTIV blue decolourization yield equal to 98.2% ± 2.7, which was in close agreement with the predicted response value.

A noticeable difference was observed between the UV-vis and FTIR spectra of BEZACTIV blue S-Matrix 150 before and after G2 treatment (Figures 3a,3b). Comparison of FTIR spectrum of control dye with extracted metabolites from Pseudomonas strain after complete decolourization clearly indicated the degradation of dye BEZACTIV blue S-Matrix 150 by G2. The decolourization data obtained in the present study, support the observations of Singh et al. [25] and Mishra et al. [26] who isolated laccase-producing bacteria for decolourization of textile dyes.

4.                   Conclusions

The present study highlights the exploitation of laccase producing extremotolerant bacteria as an alternative method for treatments of dye containing effluents. The potential of selected bacterium Pseudomonas resinovorans G2 in the decolourization of textile dye BEZACTIV blue S-Matrix 150 was studied and optimized using response surface methodology. Dye removal efficiency was dependent on various physicochemical parameters such as dye concentration, initial pH, ionic strength and incubation time. The degradation of the dye was proved by FTIR spectra. Treatment of model BEZACTIV blue effluent using laccase producing alkali-Halotolerant Pseudomonas resinovorans G2 could be used as environmentally friendly and cost-effective bioprocess for TWWs management. Analytical and toxicological experiments are in progress in order to evaluate the degradation mechanism and the safety of the end-products. Application of extremotolerant bacteria as single strains and/or as consortia for treatment of real textile effluents needs further investigation.

5.                   Acknowledgements

The authors acknowledge financial support from the European Union in the ambit of the project MADFORWATER (H2020, GA 688320) and the Tunisian Ministry of Higher Education and Scientific Research in the ambit of the laboratory project LR11ES31.

Figures 1(a-d): (a) Detection of laccase activity of G2 strain grown on TSA supplemented with 0.2% DMP, 3% NaCl at pH8 for 3 days incubation at 37^°C; (b) Bezactiv Blue decolourization and (c) Tubantin brown decolourization by G strain grown on TSB supplemented with 100 mg/L of the dye, during 2 days of incubation.

Figure 2: Contour plots and response surfaces curves showing interactive effect of dye concentration and incubation time (a) as well as pH and NaCl concentration (b) on the decolourization of Bezactiv Blue S-Matrix 150 by G2 strain.

Figures 3(a-b): The variation in UV-Vis (a) and FTIR (b) spectra of Bezactiv Blue S-Matrix 150 before and after decolourization by G2 strain.

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