Research Article | Volume 12, Issue 3, May, 2024

Sustainable biodegradation of textile dye reactive blue 222 by the novel strain Enterobacter CU2004, isolated from the industrial waste: A design of experiment based optimization study and characterisation of metabolites

Vasantha Veerappa Lakshmaiah Anish Nag Suresh Gotekar Sunil More Shobha K. Jayanna   

Open Access   

Published:  Apr 20, 2024

DOI: 10.7324/JABB.2023.70862
Abstract

Reactive Blue 222 (RB222) is widely used in textile industries and hence a common recalcitrant pollutant in the industrial effluent. Bioremediation of this dye is of significance as its one of the complex dyes with high molecular weight. In the present study, we isolated a novel bacterial strain Enterobacter CU2004 from the industrial waste and characterize using16S rRNA gene sequencing. Its potential to dye degradation was evaluated in a simple minimal salt media with the parameters namely dye concentration (100–1000 ppm), pH (4–9), temperature (15–55°C), Carbon source (Lactose, Sucrose, Glucose, Starch, and Fructose), and Nitrogen source (Casein, Yeast extract, Peptone, Tryptone, Ammonium sulphate, and Urea) in a 24 h culture. Finally, data obtained were extended to design of experiment based optimization for the degradation efficacy of Enterobacter CU2004 and to validated design space was established. The novelty is in optimizing the design space parameters for highest percentage of degradation ≥90% by the bacterial isolate Enterobacter CU2004 were finalized as 30–37°C temperature, 133–249 ppm dye concentration, Lactose as Carbon source, Yeast extract as Nitrogen source, and the pH as 8. Microbial dye degradation was confirmed by FTIR, HPLC and GCMS studies. Further studies revealed the dye intermediates and the potential of Enterobacter CU2004 toward the degradation of complex, high molecular weight industrial dye RB222.


Keyword:     Bioremediation Biodegradation Reactive blue 222 Enterobacter Design of experiment Statistical modeling


Citation:

Lakshamaiah VV, Nag A, Gotekar S, More S, Jayanna SK. Sustainable biodegradation of textile dye reactive blue 222 by the novel strain Enterobacter CU2004, isolated from the industrial waste: A design of experiment based optimization study and characterisation of metabolites. J App Biol Biotech. 2023. Online First. https://doi.org/10.7324/JABB.2023.70862

Copyright: Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike license.

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1. INTRODUCTION

Anthropogenic activities of humans are adversely affecting the environment. One such activity is the mass production and utilization of synthetic dyes [1]. Various industries use synthetic dyes as raw materials and release the unused ones into industrial effluents. Industries such as printing, pharmaceutical, food, and textile are the major consumers of these dyes [2]. As the demand for these dyes increases, their production has also increased over the past few decades. In 2020, India produced 191 thousand metric tons of dyes and is considered one of the leading producers manufacturing 80.000 products [3]. Nearly 10,000 dyes and pigments are now available for textile industries [4]. Textile industries are the leading consumers of these dyes and release unused dyes frequently at a 15% concentration into water bodies [5-7] Globally, it is estimated that approximately 280,000 tons of textile dyes are discharged into water bodies annually in the untreated form [8,9]. Removal and degradation of these dyes in the industrial effluent is one of the major environmental challenges and therefore, gaining more attention over the past few decades [10,11]. Even at a lower concentration of 1 ppm, these dyes can affect the nature and ecosystem of water. These dyes can block sunlight penetration and affect the photosynthetic process of aquatic plants [12]. Furthermore, it can lead to low dissolved oxygen levels and an increase in pH, BOD, and COD of water [13,14]. Industrial dyes are recalcitrant xenobiotics and thus tend to persist for a long time in the environment. Studies have also proved that they seriously affect all living forms as they are carcinogens, mutagens and teratogens. When humans are continuously exposed to these dyes, it can lead to health issues such as bladder cancer and skin allergy [15,16].

Among the industrial dyes, reactive dyes are most extensively used as they make the staining process is simple and cost-effective. These dyes contain reactive groups that can form a covalent bond with the fabric materials and thus resist wash fastness and lightfastness. Reactive dyes are used in dying of cellulose and polyamide materials in textile industries and, this makes reactive dyes as the one of the most common pollutants in industrial effluent [17-19]. Reactive dyes exhibit high solubility in water with varying complex chemical structures such as reactive blue 19 and reactive black 5 [20].

Microbial bioremediation is widely accepted as they catalyze degradation and complete mineralization of dyes at a faster rate than the conventional methods [21,22]. However, physiochemical methods are expensive, less efficient, and produce secondary sludge. Physiochemical methods such as biosorption by dead biomass of Rhizopus arrhizus [23] and basidiomycetes immobilized in Luffa cylindricall for RB222 were not much effective as they relied on passive adsorption principle rather than degradation of dyes [18]. Enterobacters, are gram-negative and natural inhabitant of the digestive tract in warm-blooded animals, are the most prevalent microbes in sewage waste. These are facultative microbes capable of degrading dyes at aerobic and anaerobic conditions [24,25]. Another significant feature of this isolate is forming a natural biofilm, enhancing its biodegradation ability. This natural biofilm can degrade higher concentrations of dyes and withstand adverse conditions otherwise not possible by discrete microbes [26]. Hence, Enterobacter sps may be considered as the most feasible and eco-friendly bio-remediator for reactive dye biotransformation to nontoxic end products [27,28].

The dye degradation capabilities of Enterobacter sps were reported elsewhere, majorly for Azo dyes [29]. Nevertheless, reports on applying this bacterial strain for bioremediation of Reactive Blue dye 222 are not reported till date to the best of our knowledge. RB222 is one of the most common dyes used in Indian textile industries. It is a highly recalcitrant dye due to its high molecular weight (1357.49 g mol-1) and further complicated and complex with two dia azo-groups which poses a problem in its uptake by the microbe bioremediation. Although other chemical methods are available for the degradation of this dye; however, due to its structural complexity, the microbial degradation of RB222 is challenging [30,31]. The key advantages of using this novel isolate are its ability to form biofilm and hence can resist and thrive at adverse condition and facilitates complete mineralization of RB222. Furthermore, this isolate can make the entire process of bioremediation of RB222 in industries as economical and eco-friendly since Enterobacter sp. CU2004 are facultative anaerobes showed its best performance under static conditions without any need for aeration or agitation. The isolate was able to metabolite RB222 dye in simple media without carbon or nitrogen source; however, their efficiency improved with cometabolism of carbon and nitrogen source.

Present work emphasis, the novelty in optimizing various physical and chemical parameters (pH, temperature, agitation, carbon, and nitrogen sources) through multivariate design of experiments (DOE) for the new isolate Enterobacter sp. CU2004 from the textile industrial waste to completely mineralize the RB222. Studies provide design space and knowledge on dye degradation pathway.


2. MATERIALS AND METHODS

2.1. Chemicals and Reagents

Reactive Blue 222 (RB222) dye (Praasa Overseas, India), minimal salt media (MSM) broth (M1253) comprising of Sodium chloride (2.5 g l-1), Disodium phosphate (33.5 g L-1), Potassium phosphate (15 g L-1) and Ammonium chloride (5 g L-1) used in dye degradation studies was procured from Hi-Media Laboratories (Mumbai, India), peptone, yeast extract, tryptone, and agar (Bacteriological grade, Himedia, India). Glucose, sucrose, lactose, fructose, starch, urea, ammonium sulfate, methanol, and ethyl acetate chemicals were from purchased from SRL Ltd., India.

2.2. Isolation and Screening of the Microorganisms

Waste water sludge (collected from Peenya industrial area, Bangalore, India) was inoculated into the MSM (10% v/v) and incubated with 100 ppm RB222 on an orbital shaker at 37°C, 120 rpm for 24 h. Further sub-culturing was performed at a high concentration of dye (200 ppm) with similar conditions and inoculum strength (10%). The final subculture was performed with 500 ppm dye. Bacterial strains capable of utilizing the dye were screened by serial dilution and spread plated on MSM agar supplemented with 100 ppm dye. We observed five colonies with prominent dye degradation properties and maintained them as a pure culture on MSM agar with 50 ppm dye for further studies. These isolated bacterial strains were cultured with 100 ppm dye concentration in MSM broth and incubated in an orbital shaker at 37°C, 120 rpm for 24 h. Cultures were centrifuged at 8000 rpm for 5 min, and absorbance of the supernatant was measured at 615 nm using UV–Visible spectrophotometer (Shimadzu UV).

Percentage of degradation by each isolate was estimated using the formula [32]

Percentage Dye Degradation = %PDD

Ac (Absorbance control): Media + Dye (100 ppm)

Ai (Absorbance inoculated): Media + Dye (100 ppm) + Bacteria

Bacterial isolate (B1) with the highest decolourization efficiency was selected for further studies.

2.3. Identification of Bacterial Isolate

For identification of the best performing bacterial isolate B1, it was outsourced to Barcode Biosciences, Bangalore, India. Purified genomic DNA of the bacterial isolate was subjected to 16S rRNA sequencing using BDT v3.1 kit and ABI 3730 × l Genetic Analyzer through Sanger’s di-deoxy chain termination method. Finally, the bacterial strain was identified by using Bioinformatics tools, namely, NCBI BLAST and MEGA X [33]. The model of sequence evolution used was a maximum composite likelihood, and for rate variation, the gamma distribution of 1 was used.

2.4. Univariate Study

Univariate experiments one factor at a time (OFAT) were initially performed to assess the dye degradation potential of the identified isolate (B1) at different physical and chemical conditions in an aqueous medium. Various experimental conditions such as pH (4–9), temperature (16–55°C), inoculum size (0.5–4% w/v), dye concentrations (100–1000 ppm), 1% w/v Carbon (glucose, sucrose, lactose, fructose, and starch), and nitrogen (casein, yeast extract, tryptone, peptone, urea, and ammonium sulfate) sources were maintained in individual sets in liquid culture media (MSM broth). Cultures were incubated at 37°C under static conditions except for temperature and aeration studies. PDD was calculated as per the formula mentioned earlier. All the experiments were performed as triplicates, and the results are expressed as mean ± SE (Standard error). The data were analyzed statistically by One-way analysis of variance (ANOVA) (IBM SPSS Statistics software V21.0). The statistical significance level was set as P ≤ 0.05.

2.5. Multivariate Study DOE

A multivariate and DOE approach as per earlier studies [34] was considered to optimize the bacterial isolate mediated % degradation of the dye. DOE is a tool to optimize desirable process performance based on a robust and multivariate statistical methodology. In this study, based on univariate experiment results, five major variables namely temperature (25–37°C), RB222 concentration (100–250 ppm), pH (5–8), Carbon (Control and Lactose), and Nitrogen (Control and Yeast extract) sources were selected. A customized quadratic response surface model was adopted for this study and the experiments were conducted with 29 random runs, as obtained by the DOE software (Design-Expert 12). The experimental design is shown in Table 1. Finally, a design space was established and validated with post-process optimization run.

Table 1: Experiment details for the design of experiment setup with results (% Decolorization).

RunTemperature (°C)Dye concentration (ppm)pHCarbon sourceNitrogen sourceDecolorization (%)
128.48202.757.40LactoseYeast extract92
225.00250.008.00LactoseControl32
326.50185.505.00ControlYeast extract66
426.92100.005.00ControlControl34
532.56152.508.00LactoseControl59
628.30100.007.63ControlYeast extract70
725.00100.008.00LactoseControl63
825.00250.005.00LactoseControl50
937.00100.007.48ControlControl29
1025.00250.008.00ControlYeast extract77
1132.14100.006.43LactoseYeast extract64
1237.00100.008.00LactoseYeast extract81
1328.30100.007.63ControlYeast extract88
1437.00104.675.38ControlYeast extract41
1525.00100.005.54LactoseYeast extract48
1633.46148.005.62LactoseControl71
1737.00250.007.48LactoseControl83
1833.46148.005.62controlYeast extract69
1932.14250.006.35ControlYeast extract77
2037.00190.008.00ControlYeast extract76
2130.52227.508.00ControlControl70
2229.62227.505.41LactoseYeast extract75
2328.48202.757.40LactoseYeast extract93
2437.00250.005.00LactoseYeast extract79
2537.00184.006.59LactoseYeast extract84
2632.14250.006.35ControlYeast extract80
2737.00250.005.00ControlControl42
2825.00181.756.56ControlControl60
2925.00181.756.56ControlControl61

2.6. Dye Degradation Studies

Dye metabolites was concentrated from inoculation broth using ethyl acetate solvent at different intervals of incubation period. Organic layer was subjected to rotatory evaporation to obtain crystals of dye intermediate metabolite for FTIR, HPLC, and GCMS studies. FTIR analysis was performed in mid-IR range (400–4000 cm-1) using Shimadzu-IrSpirit 1800 with ATR accessory. FTIR peaks obtained for dye intermediate metabolites due to the action of bacteria was compared with the parental dye spectrum.

High-performance liquid chromatography (Shimadzu-LCMS 8040) was performed to examine the dye degradation with 50% methanol and 50% water as mobile phase and analytes were separated in C 18 column of length 4.6 mm with run rate of 1 mL/min. To examine the dye degradation UV detector was set at λ max of RB-222 (615 nm), and dye degraded metabolites (280 nm), respectively.

Gas chromatography-mass spectroscopy was performed to determine the metabolites produced in the course of microbial degradation of RB222 using GCMS-QP2010 model. A sample volume of 1 μL was injected and the injector temperature was maintained at 280°C. Helium was the carrier gas, flown at 1 mL min−1. The temperature of the column was set at 60°C held for 4 min, and successively increased to 200°C at a ramp rate of 10°C min−1, holding 3 min, followed by 200°C–280°C at a ramp rate of 10°C min−1, holding the temperature for 9 min. Mass spectra were obtained at 70 eV. GC–MS spectra were analyzed using the software Shimadzu post-run analysis.

2.7. Statistical Analysis

The data obtained from dye degradation studies were tested for statistical significance by comparing the means of different test conditions using one-way ANOVA. The data were considered significant if P < 0.05.


3. RESULTS AND DISCUSSION

3.1. Isolation and Screening of Microorganisms

From preliminary screening, five different strains of bacteria on the solid MSM medium and tested for their degradation potential against 100 ppm of RB222 dye by using the enrichment method as performed by [1]. After 24 h of incubation in the MSM broth, isolate B1 showed the highest dye degradation activity. Based on this data, B1 isolate was selected for further studies.

The phylogenetic tree was constructed using MEGA X software [35] to compare the isolate B1 with relevant bacteria. The new isolate showed 99% homology with Enterobacter sp. strain DMKU-RP206 and is considered a new branch in the phylogenetic tree of genus Enterobacter [Figure 1]. 16S rRNA sequencing coupled with Neighbour-Joining algorithm is a common methodology used in the literature to identify new species [36]. Previously isolated two bacterial strains capable of degrading malachite green and crystal violet were confirmed with 99% similarity to Enterobacter sp HSL99 and Enterobacter sp HSL69, respectively. Isolate RKS13 capable of degrading methylene blue revealed the closest similarity with Bacillus albus [13]. Similarly, 16S rRNA sequencing revealed that isolate T2 could decolorize reactive blue160. T2 exhibited 96% similarity to Bacillus subtilis [37]. In this study, NCBI BLASTn (https://blast.ncbi. nlm.nih.gov/Blast.cgi) search and Neighbour-Joining phylogenetic tree revealed that the newly isolated strain B1 as Enterobacter sp with 99% target sequence coverage and 100% bootstrap support. The stain name was designated as Enterobacter Sp. CU2004. The sequence was deposited to NCBI Genbank (https://www.ncbi.nlm.nih.gov/genbank/, Accession No MF125281.1).

Figure 1: Phylogenetic tree of isolate Enterobacter CU2004 (B1) using Neighbor Joining method by MEGA X.



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3.2. Bacterial Growth Kinetics and Dye Degradation

The batch culture was established with 100 ppm of RB222 dye in the MSM broth and incubated at 37°C under static conditions for 12 h with an inoculum size of 4% (v/v). Bacterial growth and dye degradation were monitored at a regular 2 h, and a growth kinetics graph was plotted [Figure 2]. It was evident from the plot that the optical density of cell suspension increased exponentially with the time when measured at 600 nm. Dye degradation also increased when examined at 615 nm at the same interval. The optical density of the cell was increased from 0.256 to 2.016, indicating microbial growth. Simultaneously degradation percentage of the dye also increased from 0.067 to 33% [38]. It showed in a similar study that degradation of methyl orange also significantly increased with the increase in optical density of Bacillus stratosphericus SCA1007 when examined during the incubation period of 0–12 h. Therefore, the result of our study established the positive correlation between microbial growth and dye degradation.

Figure 2: Growth kinetics of isolate Enterobacter CU2004 (Time vs. growth and percentage of dye degradation).



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3.3. Univariate Study

For preliminary optimization of the dye decolorization efficiency of the isolate B1 (Enterobacter Sp. CU2004), we initially performed experiments by varying OFAT. Input parameters were pH, temperature (°C), inoculum size (%, v/v), dye concentration (ppm), and Carbon- Nitrogen sources. Hydrogen ion concentration in the medium, that is, pH, generally affects the decolorization ability of any bacterial isolate. Within the studied range [pH 4–9, Figure 3a], we observed that pH 7 gave the optimum efficacy for decolorization (41.24%). The beneficial effect of neutral pH on the decolorization of bacterial isolates was studied elsewhere. Ramlan et al. [39] reported optimum performance of Brevibacillus panacihumi ZBI decolorization of Reactive Blue 5 at neutral pH. Jirasripongpun et al. [40] also performed univariate studies and confirmed that Enterobacter Sp. exhibited its optimal decolorization of reactive red 195 at pH 7. Nevertheless, outcomes of these studies were limited by the fact that they were univariate in nature and effect of other parameters along with pH was not evaluated [41].

Figure 3: Graphical representation of Univariate experiments, (a) pH, (b) Temperature (°C), (c) Inoculum size (%), (d) Dye concentration (ppm), (e) Carbon source (1%) and (f) Nitrogen source (1%); All values are represented as mean ± SE; All values are statistically significant as performed by IBM SPSS software.



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In the next set of the experiment, the effect of different temperatures (15–55°C) on the degradation potential of the isolate B1 was studied. Maximal degradation (81.81%) was recorded at 37°C [Figure 3b]. Further, higher temperature like 55°C negatively affected the degradation efficacy of B1 to as low as 3.81%. Higher temperature results in loss of cell viability, denaturation of vital enzymes, and disintegration of cellular membrane. The earlier study, Roy et al. (2020) [36] showed similar observation for B. subtilis for degradation of the dye reactive blue 172. Enterobacter sp. could effectively degrade crystal violet at 35°C [42]. In general, microbes showed its promising dye degradation potential within the temperature range of 30–40°C.

Dye degradation was performed with varying inoculum sizes ranging from (0.5 to 4% v/v). Inoculum size had a direct effect on decolorization ability of isolate B1. Decolorization of RB222 dye at concentration of 100 ppm increased with increase in inoculum size [Figure 3f]. About 80% of dye decolorization was obtained with 4% (v/v) inoculum without adding carbon or nitrogen sources when incubated for 24 h. This confirms the active participation of B1 in the dye decolorization. A similar study reported improved degradation of direct blue 14 by Bacillus fermus (Kx898362) with the high inoculum size of 5% (v/v) [43].

The concentration of dye had a significant effect on dye decolorization ability of isolate. As the dye concentration was increased from 100 to 1000 ppm, the decolorization ability of the isolate B1 gradually reduced (81.51–51.53%) [Figure 3d]. Higher concentration of dye might pose a toxic effect on the microbial growth, metabolism, and saturating the active sites of the enzymes involved in dye decolorization, leading to reduced microbial efficacy [37]. noted that Bacillus sps could make maximum biofilm at low reactive red 170 dye concentration of 40 mg/ml. Mostly microbes failed to utilize azo dyes for their metabolism and growth efficiently. However, the results of the current study proved the isolate Enterobacter CU2005 could tolerate and decolorize RB222 at relatively higher concentration that that of the microorganisms reported in the literature.

Batch culture with 100 ppm of RB222 in MSM supplemented with different Carbon (Glucose, sucrose, lactose, fructose, and starch) and Nitrogen (Peptone, tryptone, caseine, yeast extract, urea, and ammonium sulfate) sources were set to study their effect on dye decolorization ability of the isolate B1. When the media was supplemented with carbon or nitrogen sources as cosubstrate, decolorization efficiency drastically increased. Lactose was found to be the ideal carbon source for showing higher percentage of dye decolorization (90%) when compared with the control, namely, without carbon source (81.5%) [Figure 3e]. Medium supplemented with yeast extract showed maximum decolorization of 69% among other nitrogen sources [Figure 3d]. Similar observations were reported elsewhere [44]. It showed an increase in the Remazol black B dye decolorization ability of Bacillus spp when the media was supplemented with the Carbon source Starch. Overall, the color removal ability of isolate B1 had greatly improved with the addition of organic carbon, while sole nitrogen supplements had less significance in the process.

3.4. DOE

DOE based multivariate experimental strategy is considered user-friendly, cost-effective, and can provide rapid results in a short span of time when compared with the conventional univariate or one-factor experimentation. Synergistic effect of a number of variables can be obtained from DOE based studies. Further, design space developed through DOE, can precisely provide effective parameter ranges to achieve expected output. The previous literature effectively utilized DOE strategy to optimize dye degradation [45,46]; however, application of such strategy for microorganism mediated dye degradation is scarce [47].

3.5. Selection of Model

The model F-value of 9.50 as determined by ANOVA implied that the model selected was statistically significant (P < 0.00001). There was only a 0.01% chance that an F-value this large could occur due to noise. The Lack of Fit F-value of 2.88 implied that it was not statistically significant relative to the pure error. There was a 12.50% chance that a lack of fit F-value this large could occur due to noise. Finally, the lack of fit was statistically insignificant (P = 0.1250). Based on this statistical analysis, a reduced quadratic model was selected. Model terms, namely, dye concentration (B), pH (C), carbon (D), and nitrogen (E) sources independently and, interactions of temperature X dye concentration (AB) and temperature X sugar (AD) were found statistically significant [Table 2]. Temperature X dye concentration (AB) was selected as interaction partners for further evaluation. The empirical relation between % degradation and factors was formed as: %degradation = 74.34 + 1.51A + 6.51B + 8.24C + 4.23D + 8.61E + 6.82AB + 6.68AD + 4.63CE + −8.48A2–8.57B2.

Table 2: ANOVA for the decolorization of RB222 by Enterobacter Sp. CU2004.

SourceSum of squaresdfMean squareF-valueP-valueLevel of significance
Model7595.2210759.529.50<0.0001Significant
A-Temperature38.37138.370.48010.4972
B-Dye concentration771.301771.309.650.0061Significant
C-pH1078.3311078.3313.490.0017Significant
D-Sugar499.891499.896.250.0223Significant
E-Nitrogen source1964.1111964.1124.570.0001Significant
AB515.181515.186.450.0206Significant
AD698.751698.758.740.0084Significant
CE340.331340.334.260.0538significant
353.041353.044.420.0499
350.081350.084.380.0508
Residual1438.781879.93
Lack of fit1269.281397.642.880.1250Not significant
Pure error169.50533.90
Cor total9034.0028

ANOVA: Analysis of variance

ANOVA for the decolorization of RB222 by Enterobacter Sp. CU2004.

3.6. Comparative Contour Plots

Comparative two-dimensional contour plots [Figure 4] comprising the global range (29–93%) of the response (% degradation of dye) aided in the selection of the other terms (D-Control/Lactose, E-Control/Yeast extract, and pH), along with the interaction term (AB). The result of the comparative contour plots indicated the higher decolorization rate in the lactose and yeast extract conditions when compared with the control at pH 8.

Figure 4: Two-dimensional contour plots of RB222 degradation by Enterobacter Sp. CU2004 as generated by design expert software for the process parameters (control, lactose, yeast extract and pH 8).



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3.7. Establishment of Design Space and Finalizing Input Parameter Ranges

In this optimization experiment, we attempted to establish design space considering a cutoff of minimum 90% decolorization response and covering a maximum range of input parameters to achieve the response target. As shown in the Figure 5, design space parameters for the ≥90% degradation was established as 30–37°C temperature, 133–249 ppm dye concentration, lactose as carbon source, and yeast extract as nitrogen source, with the pH to be maintained at 8.

Figure 5: Design space for the optimized process parameters to degrade RB222 by Enterobacter Sp. CU2004.



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3.8. Validation of the Established Design Space

A validation experiment of the established design space was performed by randomly selecting parameter values from the design space ranges. Selected parameters values were 32°C, 121 ppm dye concentration, carbon source as lactose, nitrogen source as yeast extract and pH 8. The predicted mean, as determined by the software, was 91.23%. Three replicas were run, maintaining the mentioned parameters and result was found as 93.12 ± 0.3%. The result was well within the range of ± 10%, from the predicted mean. Hence, the design space was established for degradation of RB222 by our isolate Enterobacter Sp. CU2004.

3.9. Identification and Characterization of Dye Metabolite

FTIR spectrum of parental dye (A) was compared with dye metabolites obtained at two different time intervals 24 h (B) and 72 h (C) Figure 6. The peaks of A at functional group region of FTIR shows peaks for O-H stretching found at 3400 cm-1. The range of 1600–1500 cm-1 shows the presence of alkene C=C stretching. Then, the peaks can be seen in the fingerprint region giving us clear indication of the functional groups. Range from 1500 to 1200 cm-1 shows aromatic C-C stretching, alkane C-H bending, and rocking. Range from 1200 to 1000 cm-1 shows alkane C-H wagging and aliphatic amine C-N stretching. Range from 1000 to 500 shows alkene C-H bending. Focusing on the spectrum of B, there are new peak formations and few peak deletions. The main peak deletion occurred at 3400 which was the O-H stretching peak. New peaks formed at 2900–2700 cm-1 range shows aromatic C-H stretching and alkane C-H stretching. The peak at 11640 cm-1 indicates the presence of alkene C=C stretching. The peak at 1100 shows the presence of aliphatic amine C-N stretching. The peak at 830 cm-1 shows the presence of ring C-C stretching and CH2 rocking. Range from 600 to 400 cm-1 shows the presence of ring C-C vibrations and alkane C-H bending. The spectrum of C has all the peaks similar to spectrum B except for a new peak at 1150 showing the presence of aliphatic C-N stretching and a missing peak at 620 cm-1. This shows that after degradation, there is a change in the molecular chemistry of the parental dye which is evident from the FTIR comparison spectrum [48,49].

Figure 6: FTIR spectrum of (a) RB222 (Parental dye), (b) Dye metabolites at 24 h and (c) Dye metabolites at 72 h.



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HPLC studies of the dye intermediates and the parental dye were compared with respect to their UV-Visible absorbance and retention time. Parental dye RB222 being chromogenic substance showed single peak with absorbance peak at 615 nm [Figure 7]. Microbial degraded RB222 was colorless and did not show any peak in the visible range while multiple new peaks with different retention time was observed at 254 nm [Figure 8]. This clearly states that parental dye was degraded by the isolate to different non-chromogenic metabolites [50].

Figure 7: HPLC chromatogram showing prominent single peak for Reactive blue 222 at 615 nm.



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Figure 8: HPLC elute profile of reactive blue dye metabolites at 254 nm.



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GCMS analysis of dye metabolites revealed multiple peaks obtained due to fragmentation of parental dye. The compounds and its structures were determined based on (m/z) values and its biodegradation pathway was proposed [Figures 9 and 10]. High molecular weight parental dye was degraded by Enterobacter CU2004 to lower molecular weight intermediates compounds. Initially the diazo dye undergoes asymmetric oxidative cleavage to 2-aminonapthalene-1,5-disulfonic acid(mol wt: 302.987), 3.4-diamino-5-hydroxynaphthalene-2.7- disulfonic acid (mol wt:333.993), and sodium 3 amino-5-((4-((4- ((2-(sulfooxy)ethyl)sulfonyl)phenyl)amino)-1,3,5-triazin-2-yl) phenyl)amino)benzenesulphonate (mol.wt:568.012). These dye intermediates on desulfonation and N-H bond cleavage resulted intermediates 6-aminonapthalene-1,5-disulfonic acid (mol wt: 302.987), 3.4-diamino-5-hydroxynaphthalene-2,7-disulfonic acid (mol wt: 333.993), and 2-((4-((1,3,5-triazin-2-yl)amino)phenyl)sulfonyl)ethyl hydrogen sulfate(mol.wt: 360.020) and sodium 3,5-diaminobenzenesulfonate (mol wt: 210.183). Fragmentation of the intermediates progress with desulfonation, N-H bond cleave and opening of rings to form 6-aminonapthalene-1-sulfonic acid(mol wt: 223.030), 3,4-diamino-5-hydroxynapthalene-2-sulfonic acid(mol wt: 254.036), 1,3,5-triazine(mol wt: 81.078), and 2-((4-aminophenyl)sulfonyl)ethyl hydrogen sulphate (mol wt: 281.297). These intermediates further fragmented to napthalen-2 amine(mol wt: 143.073), 7,8-diaminonaphthalen-1-ol(mol wt: 174.079), 1,3,5, triazine (mol wt: 81.078), 2-((4-aminophenyl)sulfonyl)ethyl hydrogen sulfate (mol wt: 281.297), and benzene-1,3-diamine(mol.wt 108.144). sequential reactions of deamination and dihydroxylation can fragment the compounds to smaller compounds like benzene, naphthalene enters gentisic acid pathway channelised to TCA cycle to completely mineralize the dye [51].

Figure 9: Proposed metabolic pathway of Reactive blue 222 by Enterobacter CU2004.



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Figure 10: Gas chromatography and mass spectroscopy of degraded Reactive blue 222 by Enterobacter CU2004.



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4. CONCLUSION

Degradation of most widely used complex reactive blue 222 is considered to be challenge at industrial scale; however, application of physicochemical techniques is proven to be not feasible and effective. This research identifies novel indigenous facultative microorganism Encterobacter Sp indigenous microbe in industrial waste. Optimizing the physical and chemical parameters by DOE approach had improved dye degradation efficiency to >90%. Our study revealed that the Bioaugmentation of Encterobacter Sp, CU2004 could efficiently decolorize the complex industrial dye RB222 (>90%) with the optimized parameters, namely, pH 8, 30–37°C temperature, 133–249 ppm dye concentration, lactose as carbon source, and yeast extract as nitrogen source. Successful establishment of design space further emphasizes the optimized process is scalable and therefore, can be extended to industrial scale. Considering the immense scope in the field of developing cost-effective, efficient, and scalable microbial dye degradation process, this isolate is a promising candidate in the field of Bioremediation.


5. ACKNOWLEDGMENT

I would like express my sincere gratitude to Christ (Deemed to be University), friends, and family members for their kind support in accomplishment of the research project.


6. AUTHORS CONTRIBUTIONS

All authors contributed to the study, conception, and design. Experiments were performed by Vasantha Veerappa Lakshmaiah. Anish Nag designed the DOE part of the work. Manuscript writing and final formatting were performed by Vasantha Veerappa Lakshmaiah and Anish Nag. First draft of the manuscript was reviewed and suggestions by Sunil S more and Shobha K Jayanna conceptualized and supervised the work. Suresh had verified the data generated. All authors read and approved the final manuscript.


7. FUNDING

There is no funding to report.


8. ETHICAL APPROVALS

This study does not involve experiments on animals or human subjects.


9. CONFLICTS OF INTEREST

The authors report no financial or any other conflicts of interest in this work.


10. DATA AVAILABILITY

All the data obtained in the study are represented as table or figures.


11. USE OF ARTIFICIAL INTELLIGENCE (AI)-ASSISTED TECHNOLOGY

The authors declares that they have not used artificial intelligence (AI)-tools for writing and editing of the manuscript, and no images were manipulated using AI.


12. PUBLISHER’S NOTE

All claims expressed in this article are solely those of the authors and do not necessarily represent those of the publisher, the editors and the reviewers. This journal remains neutral with regard to jurisdictional claims in published institutional affiliation.


REFERENCES

1.  Khan S, Joshi N. Molecular identification of dye degrading bacterial isolates and FT-IR analysis of degraded products. Environ Eng Res 2020;25:561-70. [CrossRef]

2.  Ihsanullah I, Jamal A, Ilyas M, Zubair M, Khan GK, Atieh MA. Bioremediation of dyes:Current status and prospects. J Water Process Eng 2020;38:101680. [CrossRef]

3.  Berradi M, Hsissou R, Khudhair M, Assouag M, Cherkaoui O, El Bachiri A, et al. Textile finishing dyes and their impact on aquatic environs. Heliyon 2019;5:e02711. [CrossRef]

4.  Srivastava A, Shukla S, Jangid NK, Srivastava M, Vishwakarma R. World of the dye. In:Research Anthology on Emerging Techniques in Environmental Remediation. Hershey, Pennsylvania:IGI Global;2022. 493-507. [CrossRef]

5.  Rai HS, Bhattacharyya MS, Singh J, Bansal TK, Vats P, Banerjee UC. Removal of dyes from the effluent of textile and dyestuff manufacturing industry:A review of emerging techniques with reference to biological treatment. Crit Rev Environ Sci Technol 2005;35:219-38. [CrossRef]

6.  Bayoumi MN, Al-Wasify RS, Hamed SR. Bioremediation of textile wastewater dyes using local bacterial isolates. Int J Curr Microbiol Appl Sci 2014;3:962-70. [CrossRef]

7.  Yang C, Li L, Shi J, Long C, Li A. Advanced treatment of textile dyeing secondary effluent using magnetic anion exchange resin and its effect on organic fouling in subsequent RO membrane. J Hazard Mater 2015;284:50-7. [CrossRef]

8.  Hsueh CC, Chen BY, Yen CY. Understanding effects of chemical structure on azo dye decolorization characteristics by Aeromonas hydrophila. J Hazard Mater 2009;167:995-1001. [CrossRef]

9.  Slama HB, Bouket AC, Pourhassan Z, Alenezi FN, Silini A, Cherif-Silini H, et al. Diversity of synthetic dyes from textile industries, discharge impacts and treatment methods. Appl Sci 2021;11:6255. [CrossRef]

10.  Ong SA, Ho LN, Wong YS, Pakri KA. Comparative study on the biodegradation of mixed remazol dyes wastewater between integrated anaerobic/aerobic and aerobic sequencing batch reactors. Rend Lincei 2017;28:497-501. [CrossRef]

11.  Pipil H, Yadav S, Chawla H, Taneja S, Verma M, Singla N, et al. Comparison of TiO2 catalysis and Fenton's treatment for rapid degradation of Remazol Red Dye in textile industry effluent. Rend Lincei Sci Fis Nat 2022;33:105-14. [CrossRef]

12.  Ajaz M, Shakeel S, Rehman A. Microbial use for azo dye degradation-a strategy for dye bioremediation. Int Microbiol 2020;23:149-59. [CrossRef]

13.  Kishor R, Saratale GD, Saratale RG, Ferreira LF, Bilal M, Iqbal HM, et al. Efficient degradation and detoxification of methylene blue dye by a newly isolated ligninolytic enzyme producing bacterium Bacillus AlbusMW407057. Colloids Surf B Biointerfaces 2021;206:111947. [CrossRef]

14.  Shrivastava R, Singh NK. Assessment of water quality of textile effluent and its treatment by using coagulants and plant material. Mater Today Proc 2021;43:3318-21. [CrossRef]

15.  Hatch KL, Motschi H, Maibach HI. Textile-dye and colored-textile allergic contact dermatitis. Exog Dermatol 2003;2:206-9. [CrossRef]

16.  Mani S, Bharagava RN. Textile industry wastewater:Environmental and health hazards and treatment approaches. In:Recent Advances in Environmental Management. United States:CRC Press;2018. 47-69. [CrossRef]

17.  Lewis DM. Developments in the chemistry of reactive dyes and their application processes. Coloration Technol 2014;130:382-412. [CrossRef]

18.  Vitali VM, Ballaminut N, Matheus DR. Decolorization of CI Reactive Blue 222 by immobilized basidiomycetes in response to different carbon and nitrogen inputs. Hoehnea 2018;45:352-60. [CrossRef]

19.  Sani ZM, Dalhatu AS, Baha'uddeen S. Bioremediation of some reactive dyes commonly used in fabric re-dyeing by Chlorella vulgaris. Methodology 2019;15:10-9. [CrossRef]

20.  Rashtbari Y, Afshin S, Hamzezadeh A, Abazari M, Poureshgh Y, Fazlzadeh M, Application of powdered activated carbon coated with zinc oxide nanoparticles prepared using a green synthesis in removal of Reactive Blue 19 and Reactive Black-5:Adsorption isotherm and kinetic models. Desalination Water Treat 2019;179:354-67. [CrossRef]

21.  Varjani S, Rakholiya P, Ng HY, You S, Teixeira JA. Microbial degradation of dyes:An overview. Bioresour Technol 2020;314:123728. [CrossRef]

22.  Kajla S, Nagi GK, Kumari R, Microorganisms employed in the removal of contaminants from wastewater of iron and steel industries. Rend Lincei Sci Fis Nat 2021;32:257-72. [CrossRef]

23.  Saraf S, Vaidya VK. Elucidation of sorption mechanism of R. arrhizusfor reactive blue 222 using equilibrium and kinetic studies. J Microb Biochem Technol 2016;8:236-46. [CrossRef]

24.  Sudha M, Bakiyaraj G, Saranya A, Sivakumar N, Selvakumar G. Prospective assessment of the Enterobacter aerogenesPP002 in decolorization and degradation of azo dyes DB 71 and DG 28. J Environ Chem Eng 2018;6:95-109. [CrossRef]

25.  Govindarajan RK, Mathivanan K, Rameshkumar N, Shyu DJ, Krishnan M, Kayalvizhi N. Purification, structural characterization and biotechnological potential of tannase enzyme produced by Enterobacter cloacaestrain 41. Process Biochem 2019;77:37-47. [CrossRef]

26.  Maurya A, Kumar R, Singh A, Raj A. Investigation on biofilm formation activity of Enterococcus faeciumunder various physiological conditions and possible application in bioremediation of tannery effluent. Bioresour Technol 2021;339:125586. [CrossRef]

27.  Nazari N, Kashi FJ. A novel combination of immobilized Enterococcus casseliflavussp. nov. with silver nanoparticles into a reusable matrix of Ca-Alg beads as a new strategy for biotreatment of Disperse Blue 183:Insights into metabolic characterization, biotoxicity, and mutagenic properties. J Environ Manage. 2023;325:116578. [CrossRef]

28.  Thangaraj S, Bankole PO, Sadasivam SK. Microbial degradation of azo dyes by textile effluent adapted, Enterobacter hormaecheiunder microaerophilic condition. Microbiol Res 2021;250:126805. [CrossRef]

29.  Al-Ansari MM, Li Z, Masood A, Rajaselvam J, Decolourization of azo dye using a batch bioreactor by an indigenous bacterium Enterobacter aerogenesES014 from the waste water dye effluent and toxicity analysis. Environ Res 2022;205:112189. [CrossRef]

30.  Yuan C, Li L, Sun Y, Wang B, Xu H, Wang Y. Treatment of reactive blue 222 wastewater by zero-valent aluminum reduction. Res Environ Sci 2016;29:1067-74.

31.  Shokoohi R, Torkshavand Z, Mahmoudi MM, Behgoo AM, Ghaedrahmati E, Hosseini FM. Effective removal of Azo dye reactive blue 222 from aqueous solutions using modified magnetic nanoparticles with sodium alginate/hydrogen peroxide. Environ Prog Sustain Energy 2019;38:S205-13. [CrossRef]

32.  Palanivelan R, Rajakumar S, Ayyasamy PM. Effect of various carbon and nitrogen sources on decolorization of textile dye remazol golden yellow using bacterial species. J Environ Biol 2014;35:781-7.

33.  Kumar S, Stecher G, Tamura K. MEGA7:Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 2016;33:1870-4. [CrossRef]

34.  Choudhuri I, Khanra K, Maity P, Patra A, Maity GN, Pati BR, et al. Structure and biological properties of exopolysaccharide isolated from Citrobacter freundii. Int J Biol Macromol 2021;168:537-49. [CrossRef]

35.  Prasad SS, Aikat K. Study of bio-degradation and bio-decolourization of azo dye by Enterobactersp. SXCR. Environ Technol 2014;35:956-65. [CrossRef]

36.  Roy DC, Biswas SK, Sheam MM, Hasan MR, Saha AK, Roy AK, et al. Bioremediation of malachite green dye by two bacterial strains isolated from textile effluents. Curr Res Microb Sci 2020;1:37-43. [CrossRef]

37.  Barathi S, Aruljothi KN, Karthik C, Padikasan IA. Optimization for enhanced ecofriendly decolorization and detoxification of Reactive Blue160 textile dye by Bacillus subtilis. Biotechnol Rep (Amst) 2020;28:e00522. [CrossRef]

38.  Akansha K, Chakraborty D, Sachan SG. Decolorization and degradation of methyl orange by Bacillus stratosphericusSCA1007. Biocatal Agric Biotechnol 2019;18:101044. [CrossRef]

39.  Ramlan MA, Azizan NA, Han BH, Kim LC, Mohamed SE, Ibrahim Z. Decolourisation of reactive Black 5 by azoreductase produced by Brevibacillus panacihumiZBI. Jurnal Teknol 2012;59:11-6. [CrossRef]

40.  Jirasripongpun K, Nasanit R, Niruntasook J, Chotikasatian B. Decolorization and degradation of C. I. Reactive Red 195 by Enterobactersp. Int J Sci Technol 2007;12:6-11.

41.  Hashem RA, Samir R, Essam TM, Ali AE, Amin MA. Optimization and enhancement of textile reactive Remazol black B decolorization and detoxification by environmentally isolated pH tolerant Pseudomonas aeruginosaKY284155. AMB Express 2018;8:83. [CrossRef]

42.  Roy DC, Biswas SK, Saha AK, Sikdar B, Rahman M, Roy AK, et al. Biodegradation of Crystal Violet dye by Bacteriaisolated from textile industry effluents. PeerJ 2018;6:e5015. [CrossRef]

43.  Neetha NJ, Chidananda B, Ujwal P, Kumar KG, Sandesh K. Optimization of Direct Blue-14 dye degradation by Bacillus fermus(Kx898362) an alkaliphilic plant endophyte and assessment of degraded metabolite toxicity. J Hazard Mater 2019;364:742-51. [CrossRef]

44.  Shah MP. Microbial degradation of textile dye (Remazol Black B) by Bacillusspp. ETL-2012. J Appl Environ Microbiol 2013;1:6-11.

45.  Mousavi M, Soleimani M, Hamzehloo M, Badiei A, Ghasemi JB. Photocatalytic degradation of different pollutants by the novel gCN-NS/Black-TiO2 heterojunction photocatalyst under visible light:Introducing a photodegradation model and optimization by response surface methodology (RSM). Mater Chem Phys 2021;258:123912. [CrossRef]

46.  Redha ZM, Yusuf HA, Burhan S, Ahmed I. Facile synthesis of ZnO nanospheres by co-precipitation method for photocatalytic degradation of azo dyes:Optimization via response surface methodology. Int J Energy Environ Eng 2021;12:453-66. [CrossRef]

47.  Ameenudeen S, Unnikrishnan S, Ramalingam K. Statistical optimization for the efficacious degradation of reactive azo dyes using Acinetobacter baumanniiJC359. J Environ Manag 2021;279:111512. [CrossRef]

48.  John J, Dineshram R, Hemalatha KR, Dhassiah MP, Gopal D, Kumar A. Bio-decolorization of synthetic dyes by a halophilic bacterium Salinivibriosp. Front Microbiol 2020;11:594011. [CrossRef]

49.  Haque MM, Haque MA, Mosharaf MK, Marcus PK. Decolorization, degradation and detoxification of carcinogenic sulfonated azo dye methyl orange by newly developed biofilm consortia. Saudi J Biol Sci 2020;28:793-804. [CrossRef]

50.  Bera SP, Tank SK. Microbial degradation of Procion Red by Pseudomonas stutzeri. Sci Rep 2021;11:3075. [CrossRef]

51.  Jain K, Shah V, Chapla D, Madamwar D. Decolorization and degradation of azo dye--reactive Violet 5R by an acclimatized indigenous bacterial mixed cultures-SB4 isolated from anthropogenic dye contaminated soil. J Hazard Mater 2012;213-4:378-86. [CrossRef]

Reference

1. Khan S, Joshi N. Molecular identification of dye degrading bacterial isolates and FT-IR analysis of degraded products. Environ Eng Res 2020;25:561-70. https://doi.org/10.4491/eer.2019.224

2. Ihsanullah I, Jamal A, Ilyas M, Zubair M, Khan GK, Atieh MA. Bioremediation of dyes: Current status and prospects. J Water Process Eng 2020;38:101680. https://doi.org/10.1016/j.jwpe.2020.101680

3. Berradi M, Hsissou R, Khudhair M, Assouag M, Cherkaoui O, El Bachiri A, et al. Textile finishing dyes and their impact on aquatic environs. Heliyon 2019;5:e02711. https://doi.org/10.1016/j.heliyon.2019.e02711

4. Srivastava A, Shukla S, Jangid NK, Srivastava M, Vishwakarma R. World of the dye. In: Research Anthology on Emerging Techniques in Environmental Remediation. Hershey, Pennsylvania: IGI Global; 2022. p. 493-507. https://doi.org/10.4018/978-1-6684-3714-8.ch026

5. Rai HS, Bhattacharyya MS, Singh J, Bansal TK, Vats P, Banerjee UC. Removal of dyes from the effluent of textile and dyestuff manufacturing industry: A review of emerging techniques with reference to biological treatment. Crit Rev Environ Sci Technol 2005;35:219-38. https://doi.org/10.1080/10643380590917932

6. Bayoumi MN, Al-Wasify RS, Hamed SR. Bioremediation of textile wastewater dyes using local bacterial isolates. Int J Curr Microbiol Appl Sci 2014;3:962-70. https://doi.org/10.1155/2014/863272

7. Yang C, Li L, Shi J, Long C, Li A. Advanced treatment of textile dyeing secondary effluent using magnetic anion exchange resin and its effect on organic fouling in subsequent RO membrane. J Hazard Mater 2015;284:50-7. https://doi.org/10.1016/j.jhazmat.2014.11.011

8. Hsueh CC, Chen BY, Yen CY. Understanding effects of chemical structure on azo dye decolorization characteristics by Aeromonas hydrophila. J Hazard Mater 2009;167:995-1001. https://doi.org/10.1016/j.jhazmat.2009.01.077

9. Slama HB, Bouket AC, Pourhassan Z, Alenezi FN, Silini A, Cherif- Silini H, et al. Diversity of synthetic dyes from textile industries, discharge impacts and treatment methods. Appl Sci 2021;11:6255. https://doi.org/10.3390/app11146255

10. Ong SA, Ho LN, Wong YS, Pakri KA. Comparative study on the biodegradation of mixed remazol dyes wastewater between integrated anaerobic/aerobic and aerobic sequencing batch reactors. Rend Lincei 2017;28:497-501. https://doi.org/10.1007/s12210-017-0622-2

11. Pipil H, Yadav S, Chawla H, Taneja S, Verma M, Singla N, et al. Comparison of TiO2 catalysis and Fenton's treatment for rapid degradation of Remazol Red Dye in textile industry effluent. Rend Lincei Sci Fis Nat 2022;33:105-14. https://doi.org/10.1007/s12210-021-01040-x

12. Ajaz M, Shakeel S, Rehman A. Microbial use for azo dye degradation-a strategy for dye bioremediation. Int Microbiol 2020;23:149-59. https://doi.org/10.1007/s10123-019-00103-2

13. Kishor R, Saratale GD, Saratale RG, Ferreira LF, Bilal M, Iqbal HM, et al. Efficient degradation and detoxification of methylene blue dye by a newly isolated ligninolytic enzyme producing bacterium Bacillus Albus MW407057. Colloids Surf B Biointerfaces 2021;206:111947. https://doi.org/10.1016/j.colsurfb.2021.111947

14. Shrivastava R, Singh NK. Assessment of water quality of textile effluent and its treatment by using coagulants and plant material. Mater Today Proc 2021;43:3318-21. https://doi.org/10.1016/j.matpr.2021.02.373

15. Hatch KL, Motschi H, Maibach HI. Textile-dye and colored-textile allergic contact dermatitis. Exog Dermatol 2003;2:206-9. https://doi.org/10.1159/000076803

16. Mani S, Bharagava RN. Textile industry wastewater: Environmental and health hazards and treatment approaches. In: Recent Advances in Environmental Management. United States: CRC Press; 2018. p. 47-69. https://doi.org/10.1201/9781351011259-3

17. Lewis DM. Developments in the chemistry of reactive dyes and their application processes. Coloration Technol 2014;130:382-412. https://doi.org/10.1111/cote.12114

18. Vitali VM, Ballaminut N, Matheus DR. Decolorization of CI Reactive Blue 222 by immobilized basidiomycetes in response to different carbon and nitrogen inputs. Hoehnea 2018;45:352-60. https://doi.org/10.1590/2236-8906-49/2017

19. Sani ZM, Dalhatu AS, Baha'uddeen S. Bioremediation of some reactive dyes commonly used in fabric re-dyeing by Chlorella vulgaris. Methodology 2019;15:10-9. https://doi.org/10.9734/ajee/2021/v15i430233

20. Rashtbari Y, Afshin S, Hamzezadeh A, Abazari M, Poureshgh Y, Fazlzadeh M, Application of powdered activated carbon coated with zinc oxide nanoparticles prepared using a green synthesis in removal of Reactive Blue 19 and Reactive Black-5: Adsorption isotherm and kinetic models. Desalination Water Treat 2019;179:354-67. https://doi.org/10.5004/dwt.2020.25020

21. Varjani S, Rakholiya P, Ng HY, You S, Teixeira JA. Microbial degradation of dyes: An overview. Bioresour Technol 2020;314:123728. https://doi.org/10.1016/j.biortech.2020.123728

22. Kajla S, Nagi GK, Kumari R, Microorganisms employed in the removal of contaminants from wastewater of iron and steel industries. Rend Lincei Sci Fis Nat 2021;32:257-72. https://doi.org/10.1007/s12210-021-00982-6

23. Saraf S, Vaidya VK. Elucidation of sorption mechanism of R. arrhizus for reactive blue 222 using equilibrium and kinetic studies. J Microb Biochem Technol 2016;8:236-46. https://doi.org/10.4172/1948-5948.1000292

24. Sudha M, Bakiyaraj G, Saranya A, Sivakumar N, Selvakumar G. Prospective assessment of the Enterobacter aerogenes PP002 in decolorization and degradation of azo dyes DB 71 and DG 28. J Environ Chem Eng 2018;6:95-109. https://doi.org/10.1016/j.jece.2017.11.050

25. Govindarajan RK, Mathivanan K, Rameshkumar N, Shyu DJ, Krishnan M, Kayalvizhi N. Purification, structural characterization and biotechnological potential of tannase enzyme produced by Enterobacter cloacae strain 41. Process Biochem 2019;77:37-47. https://doi.org/10.1016/j.procbio.2018.10.013

26. Maurya A, Kumar R, Singh A, Raj A. Investigation on biofilm formation activity of Enterococcus faecium under various physiological conditions and possible application in bioremediation of tannery effluent. Bioresour Technol 2021;339:125586. https://doi.org/10.1016/j.biortech.2021.125586

27. Nazari N, Kashi FJ. A novel combination of immobilized Enterococcus casseliflavus sp. nov. with silver nanoparticles into a reusable matrix of Ca-Alg beads as a new strategy for biotreatment of Disperse Blue 183: Insights into metabolic characterization, biotoxicity, and mutagenic properties. J Environ Manage. 2023;325:116578. https://doi.org/10.1016/j.jenvman.2022.116578

28. Thangaraj S, Bankole PO, Sadasivam SK. Microbial degradation of azo dyes by textile effluent adapted, Enterobacter hormaechei under microaerophilic condition. Microbiol Res 2021;250:126805. https://doi.org/10.1016/j.micres.2021.126805

29. Al-Ansari MM, Li Z, Masood A, Rajaselvam J, Decolourization of azo dye using a batch bioreactor by an indigenous bacterium Enterobacter aerogenes ES014 from the waste water dye effluent and toxicity analysis. Environ Res 2022;205:112189. https://doi.org/10.1016/j.envres.2021.112189

30. Yuan C, Li L, Sun Y, Wang B, Xu H, Wang Y. Treatment of reactive blue 222 wastewater by zero-valent aluminum reduction. Res Environ Sci 2016;29:1067-74.

31. Shokoohi R, Torkshavand Z, Mahmoudi MM, Behgoo AM, Ghaedrahmati E, Hosseini FM. Effective removal of Azo dye reactive blue 222 from aqueous solutions using modified magnetic nanoparticles with sodium alginate/hydrogen peroxide. Environ Prog Sustain Energy 2019;38:S205-13. https://doi.org/10.1002/ep.12971

32. Palanivelan R, Rajakumar S, Ayyasamy PM. Effect of various carbon and nitrogen sources on decolorization of textile dye remazol golden yellow using bacterial species. J Environ Biol 2014;35:781-7.

33. Kumar S, Stecher G, Tamura K. MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 2016;33:1870-4. https://doi.org/10.1093/molbev/msw054

34. Choudhuri I, Khanra K, Maity P, Patra A, Maity GN, Pati BR, et al. Structure and biological properties of exopolysaccharide isolated from Citrobacter freundii. Int J Biol Macromol 2021;168:537-49. https://doi.org/10.1016/j.ijbiomac.2020.12.063

35. Prasad SS, Aikat K. Study of bio-degradation and bio-decolourization of azo dye by Enterobacter sp. SXCR. Environ Technol 2014;35:956-65. https://doi.org/10.1080/09593330.2013.856957

36. Roy DC, Biswas SK, Sheam MM, Hasan MR, Saha AK, Roy AK, et al. Bioremediation of malachite green dye by two bacterial strains isolated from textile effluents. Curr Res Microb Sci 2020;1:37-43. https://doi.org/10.1016/j.crmicr.2020.06.001

37. Barathi S, Aruljothi KN, Karthik C, Padikasan IA. Optimization for enhanced ecofriendly decolorization and detoxification of Reactive Blue160 textile dye by Bacillus subtilis. Biotechnol Rep (Amst) 2020;28:e00522. https://doi.org/10.1016/j.btre.2020.e00522

38. Akansha K, Chakraborty D, Sachan SG. Decolorization and degradation of methyl orange by Bacillus stratosphericus SCA1007. Biocatal Agric Biotechnol 2019;18:101044. https://doi.org/10.1016/j.bcab.2019.101044

39. Ramlan MA, Azizan NA, Han BH, Kim LC, Mohamed SE, Ibrahim Z. Decolourisation of reactive Black 5 by azoreductase produced by Brevibacillus panacihumi ZBI. Jurnal Teknol 2012;59:11-6. https://doi.org/10.11113/jt.v59.1571

40. Jirasripongpun K, Nasanit R, Niruntasook J, Chotikasatian B. Decolorization and degradation of C. I. Reactive Red 195 by Enterobacter sp. Int J Sci Technol 2007;12:6-11.

41. Hashem RA, Samir R, Essam TM, Ali AE, Amin MA. Optimization and enhancement of textile reactive Remazol black B decolorization and detoxification by environmentally isolated pH tolerant Pseudomonas aeruginosa KY284155. AMB Express 2018;8:83. https://doi.org/10.1186/s13568-018-0616-1

42. Roy DC, Biswas SK, Saha AK, Sikdar B, Rahman M, Roy AK, et al. Biodegradation of Crystal Violet dye by Bacteria isolated from textile industry effluents. PeerJ 2018;6:e5015. https://doi.org/10.7717/peerj.5015

43. Neetha NJ, Chidananda B, Ujwal P, Kumar KG, Sandesh K. Optimization of Direct Blue-14 dye degradation by Bacillus fermus (Kx898362) an alkaliphilic plant endophyte and assessment of degraded metabolite toxicity. J Hazard Mater 2019;364:742-51. https://doi.org/10.1016/j.jhazmat.2018.10.074

44. Shah MP. Microbial degradation of textile dye (Remazol Black B) by Bacillus spp. ETL-2012. J Appl Environ Microbiol 2013;1:6-11.

45. Mousavi M, Soleimani M, Hamzehloo M, Badiei A, Ghasemi JB. Photocatalytic degradation of different pollutants by the novel gCN-NS/Black-TiO2 heterojunction photocatalyst under visible light: Introducing a photodegradation model and optimization by response surface methodology (RSM). Mater Chem Phys 2021;258:123912. https://doi.org/10.1016/j.matchemphys.2020.123912

46. Redha ZM, Yusuf HA, Burhan S, Ahmed I. Facile synthesis of ZnO nanospheres by co-precipitation method for photocatalytic degradation of azo dyes: Optimization via response surface methodology. Int J Energy Environ Eng 2021;12:453-66. https://doi.org/10.1007/s40095-020-00380-y

47. Ameenudeen S, Unnikrishnan S, Ramalingam K. Statistical optimization for the efficacious degradation of reactive azo dyes using Acinetobacter baumannii JC359. J Environ Manag 2021;279:111512. https://doi.org/10.1016/j.jenvman.2020.111512

48. John J, Dineshram R, Hemalatha KR, Dhassiah MP, Gopal D, Kumar A. Bio-decolorization of synthetic dyes by a halophilic bacterium Salinivibrio sp. Front Microbiol 2020;11:594011. https://doi.org/10.3389/fmicb.2020.594011

49. Haque MM, Haque MA, Mosharaf MK, Marcus PK. Decolorization, degradation and detoxification of carcinogenic sulfonated azo dye methyl orange by newly developed biofilm consortia. Saudi J Biol Sci 2020;28:793-804. https://doi.org/10.1016/j.sjbs.2020.11.012

50. Bera SP, Tank SK. Microbial degradation of Procion Red by Pseudomonas stutzeri. Sci Rep 2021;11:3075. https://doi.org/10.1038/s41598-021-82494-9

51. Jain K, Shah V, Chapla D, Madamwar D. Decolorization and degradation of azo dye--reactive Violet 5R by an acclimatized indigenous bacterial mixed cultures-SB4 isolated from anthropogenic dye contaminated soil. J Hazard Mater 2012;213-4:378-86. https://doi.org/10.1016/j.jhazmat.2012.02.010

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