Achromobacter xylosoxidans strain APZ for phthalocyanine dye degradation: Chemo-metric optimization and canonical correlation analyses

Journal of Water Process Engineering 18 (2017) 73–82

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Achromobacter xylosoxidans strain APZ for phthalocyanine dye degradation: Chemo-metric optimization and canonical correlation analyses

MARK

Madhava Anil Kumara, Puvathankandy Acharath Zamanaa, Vaidyanathan Vinoth Kumarb, Palanichamy Baskaralingamc, Kadathur Varathachary Thiruvengadaravid, Thanarasu Amudhad, ⁎ Subramanian Sivanesand, a

Department of Biotechnology, Madha Engineering College, Chennai, India School of Bioengineering, Department of Biotechnology, SRM University, Chennai, India c Department of Chemistry, College of Engineering, Anna University, Chennai, India d Department of Applied Science and Technology, Alagappa College of Technology, Anna University, Chennai, India b

A R T I C L E I N F O

A B S T R A C T

Keywords: Achromobacter xylosoxidans Canonical analysis Chemo-metric Reactive Blue 38 Phthalocyanine

The degradation of a phthalocyanine dye, Reactive Blue 38 (RB38) was achieved by employing bacterium; Achromobacter xylosoxidans strain APZ from our previous study. RB38 decolorization by A. xylosoxidans strain APZ was performed by supplementing Bushnell-Haas medium (BHM) with different agricultural residues, individually. Amongst the different agricultural residues, the groundnut shell powder nourished the bacterial strain by supplementing them with considerable total organic carbon to nitrogen (TOC/N) ratio for degrading the RB38 dye. “One-factor-at-a-time” (OFAT) approach followed by response surface methodology (RSM) and canonical correlation analysis were employed to quantify the optimal parameters influencing the RB38 decolorization by A. xylosoxidans strain APZ. The canonical analysis quantified the optimal values and suggested that RB38 decolorization could be ameliorated when BHM is supplemented with 5.133 g/L groundnut shell powder at pH 7.35 and 34.64 °C. RB38 bio-degradation was catalyzed by the different oxido-reductases of A. xylosoxidans strain APZ, which led to the desulfonation. Conclusively, the results of the phytotoxicity assays confirmed the detoxified nature of the metabolites.

1. Introduction Textile industry consumes large volume of water and chemicals for dyeing and processing the fabrics [1]. The textile effluents are highly colored and their characteristics mainly depend on the different chemical constituents of the starting materials used in different dyeing steps [2]. The discharge of voluminous untreated colored effluents is aesthetically displeasing and contributes toxicity to the ecosystem [3]. The phthalocyanine dyes in the textile wastewaters are non-volatile, hydrophilic, and thermally unstable and they are impermeable through the cell membrane, making them as a recalcitrant compound [4]. The nitro-substituted sulfonates in the benzene ring are highly persistent than the un-substituted sulfonates [5]. The partial degradation of these dyes generates toxic aromatic amines in the effluent and is always considered as environmentally detrimental [6]. The different conventional physico-chemical treatments like adsorption, membrane filtration, ion exchange, coagulation/flocculation, Fenton’s oxidation, ozonation, photo-chemical degradation and electrolysis are efficient in

⁎

Corresponding author. E-mail addresses: [email protected], [email protected] (S. Sivanesan).

http://dx.doi.org/10.1016/j.jwpe.2017.06.005 Received 24 April 2017; Received in revised form 28 May 2017; Accepted 6 June 2017 2214-7144/ © 2017 Published by Elsevier Ltd.

decolorizing the dyes [7–9]. These techniques are economically unfeasible and are incapable of completely eradicating the associated problems [10,11]. Bioremediation utilizes the biological system such as plants, enzymes and microorganisms to treat the pollutants that are considered to be environmentally reliable, and alternate to the conventional decomposition [12]. Biodegradation mediated by the bacterial metabolism is an economical and effective way of treating textile wastewater as they are rapid and environmentally compatible [13]. The use of pure bacterial strains for degradation of dyes ensures reproducible observations which can be easily interpreted using the biochemical and molecular biological analyses [14,15]. The complexity of dyeing wastewater makes the bacterial cells susceptible to exhibit declined bacterial growth, thus, there is a need for supplementing the wastewater with appropriate nutrients supporting the bacterial growth and metabolism. In order to facilitate this, the solid substrates mainly agro-industrial residues are used [12,13]. These residues are eco-friendly and their involvement in solid-state

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secondly, equilibrium RB38 uptake (qe), by the residues [22]. 5.0 g/L of the individual agricultural residues in 100 mL of 100 mg/L RB38 solution was shaken at 120 rpm for 30 min at room temperature. RB38 equilibrium concentration was determined by measuring the absorbance at 594 nm of the reaction mixture at regular intervals of time. In order to measure the adsorbed RB38 concentration on agricultural residues, the solution was subjected to desorption by adding 50 mL of dimethyl sulfoxide (DMSO) with 5.0 g of adsorbed agricultural residue and kept under shaking (120 rpm) for 20 min. The solvent laden solution was filtered through Whatmann filter paper and then centrifuged at 10,000 rpm for 10 min. All experiments were performed in triplicates and the mean values were calculated. The amount of RB38 adsorbed onto the agricultural residues, qe (mg/g), was calculated using the following relationship:

fermentation of textile dyeing wastewater offers salient merits over the submerged fermentations [16]. The physico-chemical parameters influence the dye degradation and the efficacy of bio-degradation vary with respect to the metabolic profile of the bacterial cells [15,16]. Therefore, an efficient chemo-metric tool that enhancing the microbial growth and degradation must be employed for simultaneously determining the optimal conditions of the operational parameters [17,18]. RSM is a statistical approach for process optimization involving experimental design and data fitting to an empirical model [19]. The canonical correlation analysis is a multivariate statistical model facilitating the study of interrelationships between the sets of multiple dependent and independent variables [20,21]. The present study aims to acclimatize an isolated bacterial strain to the structure of RB38 present in the pre-adaptation medium and also to focus on the co-substrate screening for biodegradation. Simultaneously, to quantify the biodegradation process parameters using chemometric tools. However, to best of our knowledge, there are no reports available for ameliorating the dye degradation using canonical correlation analyses followed by RSM. The spectral and chromatographic techniques were used to reveal the mechanism of RB38 biodegradation. The study also aims to elucidate the products of RB38 biotransformation produced by oxido-reductases using mass spectral analysis with the concomitant assessment of detoxification.

(Ci − Ce)V W

qe =

(1)

where Ci and Ce are the initial and equilibrium RB38 concentrations (mg/L) respectively; V is the volume (L); and W is the mass (g) of the individual agricultural residues. 5.0 g of RB38 adsorbed agricultural residues was added individually into 250 mL Erlenmeyer flasks and they were sterilized, prior inoculation with 3.0 mL of A. xylosoxidans strain APZ for the decolorization studies. The mixture was incubated at 30 °C under shaking condition (120 rpm) and abiotic controls were always included [23]. The decolorization was quantitatively analyzed using absorbance recorded at 594 nm on Shimadzu UV-1800 spectrophotometer model (Tokyo, Japan). The average decolorization rate, ADRRB38 (μg/min), and percentage of RB38 decolorization (%D) for the initial RB38 concentration, C (mg/L), were calculated as follows,

2. Materials and methods 2.1. Chemicals and bacterial strain RB38 (C34H24CuN8Na2O6S2, molecular weight, MW = 814.24, maximum wavelength, λmax = 594 nm and the molecular structure is given in the Supplementary Materials Fig. S1), ABTS (2,2ʹ-azino-bis (3ethyl-benzothiazoline-6 sulfonic acid)), catechol and n-propyl alcohol were purchased from Sigma-Aldrich (Bangalore, India). RB38 stock solution was prepared using double distilled water and stored in the dark at room temperature. All the chemicals were of highest purity with analytical grade. The bacterial strain to be employed in this research is A. xylosoxidans strain APZ (Supplementary Materials Fig. S2), that was identified in our previous study; Kumar et al. [29] to decolorize Reactive Green 19A (RG19A).

ADRRB38 (μg/min) =

% D=

C × % D × 1000 100 × t

Initial absorbance − Observed absorbance × 100 Initial absorbance

(2)

(3)

2.4. Chemometric modeling of RB38 decolorization

2.2. Acclimation of A. xylosoxidans strain APZ

The chemo-metric optimization tool involves the evaluation of the response, ADRRB38 (μg/min), based on the different combinations of independent operational parameters using a face-centred central composite design (CCD) matrix provided by ‘Design Expert’ (Version 8.0, Stat-Ease Inc., Minneapolis, USA). The coded variables in the CCD matrix were related by the equation given below:

The acclimation of A. xylosoxidans strain APZ was performed at 37 °C under static condition using Bushnell and Hass medium (BHM) containing (g/L): MgSO4·7H2O, 0.04; K2HPO4, 1.0; CaCl2·2H2O, 0.022; FeCl3·6H2O, 0.005; NH4NO3, 1.0 and yeast extract, 0.05. The medium with increasing RB38 concentrations (100–500 mg/L) and decreasing glucose concentrations (1.0–0.1 g/L) were used. When the culture reached the exponential phase, an aliquot of the culture was transferred to the next medium containing higher RB38 concentrations (100, 200, 300, 400 and 500 mg/L). This was done until the medium contained 500 mg/L RB38 without glucose and the cells which were able to survive was considered to be acclimated and was maintained as glycerol stock at −80 °C.

Xi =

Xi − X0 δX

(4)

where X0 denotes the coded values, Xi is the centre point and δX is the step change. The chosen factors for optimization were related to the response by the quadratic equation given below:

Y = β0 + β1 X1 + β2 X2 + β3 X3 + β11 X12 + β22 X22 + β33 X32 + β12 X1 X2

2.3. Screening of co-substrate for RB38 decolorization

+ β13 X1 X3 + β23 X2 X3

(5)

where Y is the response, β0, β1, β2, β3, β11, β22, β33, β12, β13 and β23 are the regression coefficients for the intercept, linear, quadratic and interaction effects respectively, X1, X2 and X3 are the independent variables respectively. The statistical significance of the developed model was evaluated with the help of coefficient determination (R2), predicted and adjusted correlation coefficients (R2predicted & R2adjusted) and analysis of variance (ANOVA). The response surface plots were generated to understand the interaction of the independent factors and determine the optimal level of the parameters enhancing RB38 decolorization.

The screening of suitable co-substrate was done by choosing agricultural residues such as corn cob, wheat bran, rice bran, sago ‘Hampas’, sugarcane bagasse and groundnut shell. The residues were washed with distilled water individually and dried at 80 °C prior storing in the desiccators. The dried agricultural residues were powdered and designated as CCP, WBP, RBP, SHP, SBP and GNSP respectively. The screening for the best agricultural residue as the substrate for RB38 decolorization was evaluated in two aspects; firstly, by determining the ratio between the total organic carbon (TOC) and nitrogen (N) and 74

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at 500 MHz by dissolving the samples in DMSO-d6 and a 0.3 Hz line broadening was applied prior transformation and integration. High performance liquid chromatography (HPLC) elution profiles of RB38 and biotransformed metabolites were recorded using 1260 Infinity Binary pump, (Agilent Technologies, Germany) by scanning the analytes using UV–vis detector at 594 and 335 nm for RB38 and biotransformed metabolites respectively in C-18 column (300 × 7.7 mm) operated at 35 °C with mobile-phase as HPLC grade methanol at a flow rate of 1.0 mL/min for 10 min run time. TLC analyses were performed on Aluchrosep Silica Gel 60/UV254 (SDFCL, Mumbai, India) developed with methanol: ethyl acetate: n-propanol: water: acetic acid (1:2:3:1:0.2, v/v). The mass spectra of the biotransformed metabolites were recorded using Agilent 5975C (Shimadzu, Japan) using helium as the carrier gas at a flow rate 1.0 mL/min for 30 min run time.

2.5. Canonical correlation analysis The canonical correlation analysis quantifies the stationary point (XS) of the polynomial model for characterizing the response. XS can be defined as a point at which the response surface attains a maximum, minimum or saddle (mini-max) point [20]. Let the estimated form of the polynomial model Eq. (5) be written as:

yˆ = b0 + b1 X1 + b2 X2 + b3 X3. + b4 X12 + b5 X22 + b6 X32 + b7 X1 X2 (6)

+ b8 X1 X3 + b9 X2 X3 Eq. (6) can be represented by vectors and matrix as:

yˆ = b0 + x′b + x′Bx

(7) b

b

⎡ b4 7 2 8 2 ⎤ ⎡ b1 ⎤ ⎡ X1 ⎤ x = ⎢ X2 ⎥ b = ⎢ b2 ⎥ and B = ⎢ b7 2 b5 b9 2 ⎥ ⎢ ⎥ ⎢b ⎥ ⎢ X3 ⎥ ⎢ b8 2 b9 2 b6 ⎥ ⎣ ⎦ ⎣ 3⎦ ⎣ ⎦

2.8. Detoxification assessments (8)

The extent of RB38 detoxification by A. xylosoxidans strain APZ was assessed using phyto-toxicity tests at room temperature [28] on the commonly used seeds, Phaseolus mungo, Triticum aestivum and Sorghum bicolor. The seeds were placed on layers of filter paper and supplied with 5 mL of RB38 and biotransformed metabolites individually under the controlled environment. The control sets were supplied with BHM at room temperature. The phytotoxicity assessing parameters such as percentage germination, plumule and radicle lengths were measured after 7 days of observation. The chlorophyll content of the seedlings from each sets were determined spectrophotometrically at 645 and 663 nm [29,30].

where ŷ is the predicted response, x, b and B are the vectors of the independent variables, linear and quadratic terms respectively. XS of ŷ in Eq. (7) is estimated by the relation:

b XS = −B −1 ⎛ ⎞ ⎝2⎠

(9)

If the signs of all Eigen values of matrix B are positive, XS is a minimum point, if the signs of all Eigen values of matrix B are negative, XS is a maximum point and if the signs are mixed, XS is a saddle point. 2.6. Activity determination of inducible oxido-reductases

3. Results and discussion A. xylosoxidans strain APZ was grown in BHM containing agricultural residues with adsorbed RB38 or without it (control) and the decolorized solution were centrifuged at 10,000 rpm for 30 min at 4 °C. The activities of laccase, lignin peroxidase (LiP), manganese peroxidase (MnP) and azoreductase were determined spectrophotometrically in the culture supernatant at 35 °C. Laccase activity was determined by measuring the oxidation of ABTS at 420 nm [24]. LiP activity was determined by monitoring the formation of propanaldehyde at 300 nm [25]. MnP activity was determined by monitoring the oxidation of 2,6dimethoxyphenol at 469 nm [26]. Azoreductase activity was measured by monitoring the removal of methyl red at 430 nm [27]. One unit of enzyme activity is defined as a change in absorbance units/min/mg of protein. All enzyme assays were carried out at room temperature, where reference blanks contained all the components except the crude enzyme solution. All enzyme assays were run in triplicates and the mean values were obtained to calculate the enzyme activities.

3.1. Acclimation of RB38-decolorizing bacterial strain A. xylosoxidans strain APZ had the ability to show a light zone of clearance on NA plates containing RB38. The strain was further tested for their degradation capability in liquid media and the bacterial cells were acclimated using BHM with decreasing glucose and increasing RB38 concentrations. The decolorization experiments were performed under static condition to avoid the inhibition offered by the oxygen molecules during the reduction of RB38. Inoculated Erlenmeyer flasks dispensed with sterile BHM containing 100 mg/L RB38 and 1.0 g/L glucose were decolorized at a lesser time (180 min) when compared to BHM with 500 mg/L RB38 and 0.1 g/L glucose (640 min). The adaptation of the isolate was done by making the cells to employ alternate metabolic pathways for assimilating the organo-pollutant [5,31]. A. xylosoxidans strain APZ partially decolorized (i.e., 78%) 500 mg/ L RB38 in BHM without glucose after 900 min, giving an ADRRB38 of 679.68 μg/min. RB38 decolorization was mediated by the bacterial action but not with the action of pH and this was confirmed by the periodical monitoring of pH level. There was no change in pH level of the initial and final RB38 solution treated with A. xylosoxidans strain APZ. The findings from other researchers reveal that A. xylosoxidans had genes which are responsible for the biodegradation of aromatic or halogenated compounds [32,33].

2.7. Characterization of biotransformed metabolites RB38 biodegradation was monitored using the spectrophotometric, spectroscopic and chromatographic analyses. Initially, distilled water was added to the agricultural residues with adsorbed RB38 treated with A. xylosoxidans strain APZ followed by incubation at 120 rpm for 1 h. Finally, the mixture was centrifuged at 10,000 rpm for 10 min followed by extraction using the equal volume of ethyl acetate and drying. The supernatant of RB38; before and after decolorization were scanned in the region 300–900 nm using UV–vis spectrophotometer. The biotransformed metabolites and RB38 were characterized using Fourier transform infrared (FT-IR) spectra in the mid-IR region (400–4000 cm−1) using Perkin-Elmer 237B Infrared spectrometer. The samples were mixed with spectroscopically pure KBr in the ratio of 5:95 and made as pellets which were fixed in the sample holder for analyses with 16 scan speed. Proton-nuclear magnetic resonance (1H NMR) spectra initial of RB38 and biotransformed metabolites were recorded on a Bruker Avance DRX500 spectrometer (Bruker, Germany) operating

3.2. Effective utilization of agricultural residues as substrate for RB38 decolorization The presence of dyes in the wastewater would be highly toxic to the biological system as they are deficient in the essential nutrients to degrade these recalcitrant compounds. So, it is necessary to supplement the dyeing wastewater with substrates and/or co-substrates which are required for the growth of A. xylosoxidans strain APZ with the simultaneous breakdown of RB38 molecules. The agricultural residues with adsorbed RB38 nourished the bacterial strain and improved the contact between the strain and RB38, thereby facilitating an enhanced 75

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Fig. 1. (a) TOC/N ratio and qe (mg/g) for the different agricultural residues and (b) effect of agricultural residues on ADRRB38 (μg/min) by A. xylosoxidans strain APZ.

treatability [34]. TOC/N ratios were CCP, 15.28; WBP, 41.27; RBP, 37.82; SHP, 26.06; SBP, 19.79 and GNSP, 45.82. The values of qe were CCP, 1.64; WBP, 1.48; RBP, 1.54; SHP, 1.74; SBP, 1.42 and GNSP, 1.8 (mg/g). The value of ADRRB38 was 208.333 μg/min when BHM was supplemented with RB38 adsorbed on GNSP, resulting in a complete decolorization after 8 h (Fig. 1a & b). WBP had high TOC/N ratio but failed to adsorb RB38 effectively, which resulted in ADRRB38 of 69.4 μg/ min. Thus, the presence of GNSP nourished the bacterial cells and increased the decolorization.

Table 1 The observed ADRRB38 (μg/min) for the CCD matrix.

3.3. Chemometric optimization of operational parameters and regression model evaluation RB38 decolorization efficiency was observed to be 18–96 % for the different experimental combinations given by the CCD matrix (Table 1). The value of ADRRB38 was high when A. xylosoxidans strain APZ was inoculated in BHM supplemented with 4.0 (g/L) of GNSP at pH 8.0 and 37 °C for 8 h. The regression model was developed for predicting the optimal level of parameters for improved RB38 decolorization.

ADRRB38 (μg/min) = 194.72 − 4.79X1 − 6.04X2 + 13.12X3 − 41.48X12 − 24.81X22 − 14.39X32 + 12.24X1X2 − 10.16X1X3 (10)

− 6.51X2X3 2

The regression model for the rb38 decolorization had R and

R2adjusted 76

Run

pH

Temperature (°C)

GNSP (g/L)

ADRRB38 (μg/min)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

4.0 12.0 4.0 12.0 4.0 12.0 4.0 12.0 4.0 12.0 8.0 8.0 8.0 8.0 8.0 8.0 8.0 8.0 8.0 8.0

27 27 47 47 27 27 47 47 37 37 27 47 37 37 37 37 37 37 37 37

2.0 2.0 2.0 2.0 6.0 6.0 6.0 6.0 4.0 4.0 4.0 4.0 2.0 6.0 4.0 4.0 4.0 4.0 4.0 4.0

114.583 85.417 83.333 122.917 164.583 114.583 127.083 106.250 143.750 156.250 177.083 156.250 164.583 189.583 197.917 200.000 195.833 195.833 195.833 195.833

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Table 2 ANOVA for ADRRB38 (μg/min) from CCD matrix. Source

Sum of squares

Model 29294.84 X1 229.58 X2 365.01 X3 1722.63 2 4730.96 X1 2 X2 1692.80 X32 569.77 X12 1198.47 X13 825.22 X23 339.08 Residual 660.81 Lack-of-Fit 645.61 Pure error 15.19 Correlation 29955.65 Total R2 = 0.978 R2adjusted = 0.958 R2predicted = 0.7415 Standard deviation = 8.13

Degrees of freedom

Mean squares

F-value

P-value (Prob > F)

9 1 1 1 1 1 1 1 1 1 10 5 5 19

3254.98 229.58 365.01 1722.63 4730.96 1692.80 569.77 1198.47 825.22 339.08 66.08 129.12 3.04

49.26 3.47 5.52 26.07 71.59 25.62 8.62 18.14 12.49 5.13

< 0.0001 0.0919 0.0406 0.0005 < 0.0001 0.0005 0.0149 0.0017 0.0054 0.0469

42.49

0.0004

yˆ = 194.72 + x ′b + x ′Bx

(11)

⎡ X1 ⎤ X = ⎢ X2 ⎥ ⎢ X3 ⎥ ⎣ ⎦

(12)

⎡− 4.79 ⎤ b = ⎢− 6.04 ⎥ ⎣ 13.12 ⎦

(13)

− 5.08 ⎤ ⎡− 41.48 6.12 B = ⎢ 6.12 − 24.81 − 3.255 ⎥ ⎣ − 5.08 − 3.255 − 14.39 ⎦

(14)

The vectors such as b and B show that the linear, interaction and quadratic effects of pH, temperature and GNSP are significant (bii ≠ 0, matrix B). The point of maximum response was determined through canonical analysis and the value of XS was found accordingly:

⎡− 0.162 ⎤ XS = ⎢− 0.236 ⎥ ⎣ 0.5664 ⎦

PRESS = 7744.64 Mean = 154.37 CV = 5.27% SNR = 19.301

(15)

The characteristic roots (λi) of matrix B are negative (−11.92, −43.994 and −24.76) which means a maximum response. The coded value obtained for ADRRB38 from Eq. (15) was X1 = −0.162 which corresponds to pH 7.352. Similarly, the coded values for the temperature (X2 = −0.236) and GNSP concentration (X3 = 0.5664) corresponds to 34.64 °C and 5.13 (g/L) respectively, thereby confirming that the values are within the limit of the actual values as defined by the following relations as described by Myers & Montgomery, [21]:

values of 0.978 and 0.958 respectively, indicating that the developed model is highly significant and the independent variables contribute positively to the response [35,36]. The statistical significance and adequacy of the developed model were evaluated by examining the data given by ANOVA (Table 2). It is evident that the process had larger Student’s test (t) and low P-value for the linear and interaction effects of the variables [37]. It is understood from ANOVA that the variation from the model is significant and the linear effect of pH is the only statistically insignificant term which failed to produce a better response. The model had the following characteristics: F-test, 49.26; SNR, 19.301 and CV, 5.27%, thereby proving that the model is significant and adequate to explain the interactions with a greater reliability. In addition to R2 and R2adjusted, ANOVA, F-ratio and t-and P-values, the model adequacy was also evaluated in terms of the residuals which are a few points in the CCD matrix causing non-normality of the residual distribution [38]. The normal probability plot (Supplementary Materials Fig. S3a) indicates whether the residuals would follow a normal distribution which is represented as a straight line. The plot for the model prediction versus the actual observed responses (Supplementary materials Fig. S3b) indicates that the straight line describes the goodness of the linear response surface model and the randomly scattered nature of the residuals. The residuals versus run plot (Supplementary materials Fig. S4a) was more appropriate for the developed model as the plot had a horizontal band indicating that there was no defects in the regression model [37]. The plot between the predicted versus actual values (Supplementary materials Fig. S4b) represent a satisfactory correlation between the experimental and predicted responses. The cluster of points near the diagonal line in the plot explains the goodness-of-fit. Box-Cox plot for power transforms (Supplementary materials Fig. S5) were used to determine the most appropriate power transformation to idealize the slight deviation of the responses [39]. The surface plots explain the behaviour of the response with respect to the simultaneous change in the variables as shown in Fig. 2(a–f) and the optimal levels (identified as hump and smallest ellipse) in maximizing the response were determined [40]. The curvature is due to the very low P-value for the linear, quadratic and interaction terms. The response is maximized at the centre point of all the parameters investigated for enhancing RB38 decolorization.

X1 =

pH−8 4

(16)

X2 =

Temperature(°C) − 37°C 10°C

(17)

X3 =

GNSP(g/L) − 4(g/L) 2(g/L)

(18)

As a result, ADRRB38 of 194.72 (μg/min) was observed when A. xylosoxidans strain APZ was inoculated in an Erlenmeyer flask containing 100 mg/L RB38 and 5.13 g/L GNSP when incubated at pH 7.35 at 34.64 °C under static condition. 3.5. Analyses of RB38 biodegradation by A. xylosoxidans strain APZ 3.5.1. UV–vis spectrophotometric analyses The mechanism of RB38 decolorization was studied using UV–vis spectroscopy [41] and the UV–vis spectra of initial and decolorized RB38 were compared (Supplementary materials Fig. S5). It was found that the major visible absorbance peak at 594 nm of rb38 solution disappeared completely in the UV–vis spectra and the absorbance peak was found at 335 nm in case of decolorized solution. 3.5.2. FT-IR spectral analyses The functional groups in the products were determined using FT-IR spectroscopic analyses and the variations in the fingerprint region (1500–500 cm−1) of the spectra confirm that A. xylosoxidans strain APZ biodegraded RB38. FT-IR spectrum of RB38 showed a peak for the alkyl ether at 1135 cm−1 for the asymmetric R-O-R stretching. 1049 cm−1 refers to the asymmetric S]O stretching as in the case of sulfonic acids. Peaks at 3458 and 642 cm−1 represent the NeH and CS stretching, describing the sulfonic nature of RB38 (Fig. 3a). FT-IR spectrum of the bio-transformed products had a peak at 991 cm−1 for CH stretching. The peaks at 621 cm−1 with 2173 cm−1 for OeH stretch indicate the formation of high concentration of alcohols. The peaks such as 1640 and 2078 cm−1 describe NeH deformation and NH3 stretching to amino amide and a peak at 1011 cm−1 represents the CeOH stretching (Fig. 3b). The disappearance of 1049 cm−1 peak for S]O stretching in

3.4. Canonical correlation and quantification of the interrelationships XS was evaluated by performing canonical correlation analyses of the following fitted response surface model in matricial by: 77

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Fig. 2. Response surface plots for the ADRRB38 (μg/min) as a function of (a) temperature (°C) and pH; (b) GNSP (g/L) and temperature (°C); (c) GNSP (g/L) and pH.

attached to the nitrogen atom (methylene protons). The signals between 2.063 and 2.1 ppm were attributed to the OH proton of the SO3H group (Fig. 4a). 1 H NMR spectrum of the products displayed a peak at 1.839 ppm and had signals between 2.306 and 2.338 ppm for the methyl protons. The presence of CH3eC]O was represented by the signal at 2.007 ppm. The absence of peak at 7.606 ppm indicates the CeN bond rupture between the rings. The absence of the corresponding peaks for the aromatic protons in the low field zone describes RB38 degradation. A spin of network in the high field zone was observed 1.562 and

the bio-transformed products explains that RB38 did not get transformed into sulfo-derivatives [42]. 3.5.3. 1H NMR spectral analyses 1 H NMR spectrum of RB38 had peaks between 7.372 and 7.606 ppm, indicating the presence of three aromatic proton resonances in the ring. The peak at 7.606 ppm is due to the aromatic protons present in the ring carrying the sulfonate group. The singlet at 7.372 ppm is due to the proton of naphthalene and benzene ring of RB38. The signal at 3.703 ppm is attributed to the two methyl groups 78

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Fig. 3. FT-IR spectra of (a) initial RB38 and (a) metabolites.

3.5.5. Mass spectral analyses The mass spectra of the bio-transformed products displayed sixteen different compounds produced as a result of RB38 degradation (Fig. 6). RB38 was degraded into simpler products of varying MWs ranging from 334 (Rt: 15.89 min, 3,3ʹ-bis (1, 2, 4-oxadiazolyl)-5,5ʹ-diamine) to 99 (Rt: 8.53 min, 1-methyl-2-pyrrolidinone) respectively (Supplementary materials Table S1). Heinfling-Weidtmann et al. [43] bio-transformed two sulfonated dyes; RB15 and RB38 using Bjerkandera adusta and found sulfophthalimide as the product of bio-degradation. But, in the present investigation, the bio-transformed products did not possess any residues of sulfur. Thus, GC–MS analysis confirmed the removal of sulfone groups which supported the findings from FT-IR analysis. RB38 is a sulfonated dye and the elimination of sulfone groups in the treated effluent would add environmental safety benefits. The cells of A. xylosoxidans strain APZ exhibited a remarkable phenomenon in the elimination of S]O groups and this may be due to the reason that the bacterial cells would have assimilated the sulfates for the synthesis

1.626 ppm which were attributed to the saturated/unsaturated aliphatic compounds resulting from the cleavage of naphthalene and benzene rings (Fig. 4b). After RB38 degradation, only a few signals were found between 0.835 and 2.338 ppm, indicating the formation of low molecular weight aliphatic hydrocarbons such as free methyl groups.

3.5.4. Chromatographic analyses The HPLC elution profiles of RB38 and bio-transformed products described that the biodegradation of RB38 had occurred. The chromatogram of RB38 had a major peak at Rt of 0.912 min with a shoulder peak at 0.791 min, while new peaks emerged at different Rt (1.752, 1.461, 1.33, 1.068 and 0.951 min) in the case of the bio-transformed products (Fig. 5). TLC sheet showed a single band for RB38 with the retardation factor (Rf) of 0.81, while the bio-transformed products had Rf values of 0.76 and 0.63, illustrating biodegradation of RB38. 79

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Fig. 4. 1H NMR spectra of (a) initial RB38 and (a) metabolites.

of sulfur-containing cellular components by assimilatory sulfate reduction mechanism. Thus, from the economic as well as environmental point of view, A. xylosoxidans strain APZ could be a reliable mean for the technologies aiming at effluent mineralization.

extracellular enzymes were responsible for the asymmetric cleavage of the polyaromatic ring of RB38 [11]. The aforementioned intriguing observation on the involvement of enzymes indicated that RB38 decolorization was achieved by degradation, not by adsorption.

3.6. Status of oxido-reductases during biotransformation

3.7. Detoxification by A. xylosoxidans strain APZ

The activities of various oxido-reductases during A. xylosoxidans strain APZ mediated RB38 biodegradation are summarized in Table 3. Peroxidases (LiP and MnP) can catalyze biotransformation of aromatic dyes either by precipitation or by opening the aromatic ring structure [44,45]. The decolorization started after 2 h, while the activities of laccase and azoreductase were minimal which describes that the initial decolorization was not promoted by laccase and azoreductase [13]. After 6 h, the oxido-reductive cleavage was initiated by the azoreductase, whose activity was the highest after 7 h. Similarly, tyrosinase activity increased gradually during the biotransformation. These

The plant growth parameters (plumule and radicle emergence) along with the chlorophyll content of the seedlings treated with the biotransformed products were comparatively greater than that of the seedlings treated with RB38 as shown in Table 4. The biotransformed metabolites of RB38 did not exhibit any growth inhibition in terms of elongation of plumule, radicle, and the total chlorophyll content [46,47]. This may be due to the reason that A. xylosoxidans strain APZ had mineralized RB38 which fetched essential minerals for easier assimilation by the seedlings, thereby confirming the extent of RB38 detoxification. 80

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Fig. 5. HPLC profile of (a) RB38 and (b) metabolites.

Fig. 6. MS profile for the metabolites formed after RB38 degradation by A. xylosoxidans strain APZ.

4. Conclusions

Table 3 Oxido-reductases of A. xylosoxidans strain APZ during RB38 degradation. Enzymes and their activity (Units/min/mg of protein)

Laccase LiP MnP Azoreductase

The role of unexploited GNSP as a macronutrient was meritorious in utilizing the waste agricultural residues as a supporting material for nourishing the cells of A. xylosoxidans strain APZ for phthalocyanine dye degradation. The canonical analysis quantified the interrelationships of the process parameters. The sulfone groups were not found in the RB38 solutions treated with A. xylosoxidans strain APZ. The results from this study reveal that the desulfonation and detoxification of dyes would be a trustworthy application, particularly in the treatment of concentrated dyeing wastewater.

Induction 0h

8h

0.036 ± 0.011 0.039 ± 0.008 0.011 ± 0.005 0.12 ± 0.01

0.14 ± 0.02 0.082 ± 0.023 0.061 ± 0.018 0.67 ± 0.10

81

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Table 4 Phytotoxicity of RB38 and their metabolites for the P. mungo, T. aestivum and S. bicolor. No.

Treatment solution

GP (%)

Plumule length (cm)

Radicle length (cm)

[19]

Chlorophyll content (mg/g tissue)

P. mungo 1 Control RB38 Metabolites

90 80 90

9.45 ± 0.67 7.5 ± 1.3 9.9 ± 1.8

13.55 ± 1.23 3.2 ± 1.0 10.7 ± 1.3

0.075 0.011 0.068

T. aestivum 2 Control RB38 Metabolites

90 70 90

8.60 ± 0.54 5.12 ± 0.36 7.93 ± 1.39

6.35 ± 1.10 0.05 ± 0.003 1.25 ± 0.33

0.015 0.009 0.0115

S. bicolor 3 Control RB38 Metabolites

100 80 100

11.85 ± 0.21 6.9 ± 0.39 9.17 ± 0.70

6.15 ± 0.66 2.18 ± 0.38 5.17 ± 0.13

0.027 0.013 0.103

[20] [21] [22]

[23]

[24]

[25]

Appendix A. Supplementary data

[26]

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jwpe.2017.06.005.

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