Bioremediation of Brilliant Red Azo Dye Toxicity in Vigna radiata Using Encapsulated Bacillus subtilis SC9

ABSTRACT

The discharge of azo dye‐containing wastewater threatens soil microbial activity, plant growth, and ecosystem health because of the persistence and toxicity of synthetic dyes. However, the potential of immobilized siderophore‐producing bacteria for simultaneous dye decolorization and mitigation of dye‐associated phytotoxicity remains unexplored. This study highlights the decolorization and bioremediation potential of viable free cells and calcium‐alginate immobilized Bacillus subtilis SC 9 against brilliant red (BR) dye. Among ten previously identified siderophore producing bacteria (SPB), B. subtilis SC 9 exhibited the highest decolorization efficiency (88.5%). Under optimized conditions, free and immobilized cells achieved 88.5% and 94.86% decolorization, respectively, within 9 days. FTIR and GC‐MS analyses confirmed structural transformation of BR dye into simpler degradation intermediates, indicating biodegradation rather than adsorption. Greenhouse studies demonstrated that immobilized B. subtilis SC9 alleviated BR dye phytotoxicity by increasing root length (142.16%), shoot length (160.91%), soil dehydrogenase activity (192.37 µg TPF g^{− 1} soil), FDA hydrolysis (33.71 µg fluorescein g^{− 1} soil), alkaline phosphatase activity (852.20 µg pNP g^{− 1} soil), and siderophore production (331.53%) compared with untreated dye stress. Overall, calcium‐alginate immobilized B. subtilis SC 9 is a promising eco‐friendly bioinoculant for azo dye bioremediation. Future studies should validate its performance under pilot‐scale and field conditions.

Calcium alginate‐encapsulated siderophore‐producing Bacillus subtilis SC9 enhances brilliant red dye biodegradation compared with free cells. Spectroscopic analyses, specifically FTIR and GC‐MS confirmed the conversion of the dye into simpler intermediates. Concurrently, greenhouse studies indicated a reduction in phytotoxicity, an improvement in soil biological activity, siderophore production, and plant growth. These findings underscore the potential of bioinoculant for sustainable remediation of textile wastewater.

1. Introduction

Rapid industrialization has led to the increased use of dyes in several industries such as textiles, food, paper, dye, cosmetics, and leather. Several textile dyes used to color fabrics are produced annually to an estimate of approximately 7 × 10⁵ tons worldwide [1]. During the process of fabric dyeing, only 85% of the dye was bound to the fibers of the fabrics and the remaining 15% of the dyes were considered to be a waste [2]. Despite the large contribution of textile mills to economic development, these textile industries have the major drawback of a lack of proper waste management facilities. Thus, wastewater from the textile industry is either dumped into the soil of nearby areas or discharged into nearby rivers [3]. Carmen and Daniela [4] reported that during the fabric dyeing process in textile industries, 25% of the dyes are lost and 20% are discharged directly as aqueous effluent components into the soil and water environments. These dyes (methyl red, mercury orange, trypan blue, brilliant red (BR), and brilliant blue) contain azo compounds and are considered mutagenic or carcinogenic to life forms [5]. Azo dyes not only disrupt soil microbial communities but also adversely affect plant growth, nutrient availability, and crop productivity. Arshad et al. [6] reported the toxicity of Red‐S3B dye on soil microbial activities thereby affecting the growth and yield of wheat plant. Due to their complex structure and xenobiotic nature, these dyes and their products are not able to degrade completely and hence are considered toxic pollutants in textile wastewaters [7]. The removal of these textile dyes containing azo compounds or reducing the toxicity of these azo dyes is a major challenge in the textile industries.

Numerous physicochemical methods such as ozonation, adsorption, membrane filtration, radiolysis, electrochemical, and coagulation have been adopted to treat textile wastewater; however, these conventional processes are hurdled by excessive chemical consumption and high costs [8]. Conventional physicochemical methods for dye removal are often associated with high operational costs and the generation of secondary pollutants. Recent advances have also explored nanotechnology‐based approaches for dye remediation. Biogenic ZnO nanoparticles synthesized from Phyllanthus acidus have demonstrated efficient photocatalytic degradation of methylene blue dye under light irradiation, highlighting the potential of green nanomaterials for wastewater treatment. However, despite their high degradation efficiency, nanoparticle‐based methods may require external energy input and do not inherently restore soil biological functions, emphasizing the need for sustainable microbial bioremediation strategies [9]. Microorganisms including bacteria, fungi, yeasts, and algae have been investigated for dye remediation. Bacteria were selected in the present study because they generally offer rapid growth, relatively simple cultivation, diverse metabolic capabilities, and the potential to combine dye transformation with plant‐growth‐promoting traits [10]. In particular, Bacillus species are attractive because of their environmental robustness and ability to survive under variable conditions [10, 11, 12]. Bacillus subtilis has emerged as a promising bacterium for the biodegradation of textile dyes because of its rapid growth, metabolic versatility, and production of extracellular oxidoreductive enzymes involved in dye degradation. Recent studies have demonstrated that immobilization of B. subtilis within biopolymeric matrices significantly enhances bacterial stability, reusability, and decolorization efficiency under optimized conditions. In particular, chitosan‐immobilized B. subtilis has shown excellent removal of Reactive Orange 16 dye, highlighting the potential of immobilization strategies for sustainable wastewater treatment. These findings support the application of immobilized B. subtilis as an effective and environmentally friendly approach for textile dye bioremediation [13]. Consequently, microbial bioremediation has gained considerable attention as an environmentally friendly and sustainable approach, owing to the ability of microorganisms to transform or mineralize complex dye molecules under mild conditions [14, 15, 16].

To address these challenges, this study focused on the use of encapsulated siderophore producing microbes for the remediation of environmental pollutants such as BR azo dye (BR dye) effluents. Recently, siderophores have gained much attention in environmental science, as siderophores have an affinity to bind a number of metals and their chemical structures and properties [14, 15, 16]. Moreover, siderophores produced by microorganisms scavenge and transport Fe, a micronutrient required for their growth. Microbes use Fe to generate reactive oxygen species (ROS), which can attack organic molecules including dyes, resulting in the decolorization or degradation of organic molecules. Several studies have reported that the encapsulated siderophore producing microbes perform better metal sequestration than nonencapsulated pure [16, 17, 18]. Only a few have explored the role of siderophore in dye decolorization; however, it has been reported that the encapsulated siderophore producing bacteria (SPB) significantly increased decolorization efficiency and reduced reaction time for the efficient removal of dyes in the textile industries at an industrial scale [19, 20].

The encapsulated microbial cultures have several benefits over nonencapsulated or free systems. Encapsulated microbial cells have a wide range of applications including industrial and environmental scales and can be used in semi‐continuous or continuous treatment processes, thereby reducing the cost of regeneration of biocatalysts and reducing environmental pollution by degrading harmful compounds such as azo compounds. Calcium alginate was selected for encapsulation or immobilization because it is biocompatible, relatively inexpensive, nontoxic, and allows bacterial cells to be immobilized under mild conditions. The porous structure of alginate beads permits diffusion of nutrients and dye molecules while retaining viable cells, making it suitable for repeated or prolonged bioremediation applications [21, 22]. BR was selected because it is an industrially relevant synthetic azo dye and represents a persistent color pollutant whose removal and detoxification are environmentally important. In addition, the use of a commercially sourced dye provides relevance to industrial dye‐contaminated environments. Moreover, Vigna radiata was selected as a phytotoxicity model because it is a rapidly germinating, agriculturally important legume with measurable root and shoot responses, making it suitable for evaluating the effects of dye contamination and microbial remediation.

Although microbial decolorization of azo dyes has been widely investigated, several gaps still remain unanswered. First, the use of siderophore‐producing plant‐growth‐promoting bacteria in encapsulated form for simultaneous dye removal and reduction of phytotoxicity remains insufficiently explored. Second, limited studies have compared free and immobilized bacterial cells in relation to dye decolorization, plant response, soil microbial activity, and siderophore production. Therefore, the present study was designed to address these gaps. We hypothesized that encapsulation of the siderophore‐producing B. subtilis SC9 cells in calcium alginate would enhance BR dye decolorization compared with free cells and reduce the phytotoxic effects of the dye while improving microbial activity in contaminated soil. Therefore, the main aims of the present research are (i) to characterize the previously isolated SPB for BR dye decolorization efficiency and plant growth promoting traits; (ii) to optimize several parameters of bacterial growth such as nitrogen source, carbon source, temperature, and pH for efficient BR dye decolorization; (iii) to analyze the chemical composition of BR dye and the decolorized BR dye via FTIR and GC‐MS analysis; (iv) to assess the bioremediation potential of the most efficient isolate, both in free and encapsulated forms for BR dye decolorization; and (v) to evaluate the phytotoxicity of the decolorized BR dye. The novelty of this study lies in the integrated evaluation of a siderophore‐producing B. subtilis strain in both free and calcium‐alginate‐encapsulated forms for BR dye decolorization, chemical transformation, phytotoxicity reduction, and improvement of soil biological activity.

2. Results

2.1. Screening of SPB for BR Dye Decolorization

Four of the 10 SPB strains showed a clear zone on nutrient agar medium amended with 100 ppm BR dye. The aerobic decolorization of BR dye was visualized via BR dye decolorization by the four strains CWTS 10 (Bacillus albus), IPTS 7 (Rhodococcus kroppenstedtii), KBN 12 (Pseudomonas aeruginosa), and SC 9 (B. subtilis), which ranged from 27.65% to 88.5% on Day 9 (Figure 1). The maximum decolorization of the BR dye (88.5%) was observed for the strain SC 9 (B. subtilis).

FIGURE 1.

Quantitative analysis of BR dye decolorization by bacterial isolates (%) from Day 3–9. Data represented are an average of seven replicates. Data analysis was carried out by one‐way ANOVA using DMRT and treatment means (p < 0.05) were compared. The error bars represent the standard error of means of replicates. The lowercase letters above the bars denote the significance according to Duncan's Multiple Range Test (DMRT) (p ≤ 0.05).

2.2. Optimization Parameters of BR Dye Decolorization by B. subtilis SC 9

In the absence of supplementary carbon and nitrogen sources, B. subtilis SC9 exhibited only 84.67% BR dye decolorization (data not shown) after 9 days under static conditions. The effects of the carbon source, nitrogen source, pH, and temperature on BR dye aerobic decolorization (100 ppm) were evaluated after 9 days of incubation at 37°C. Among the carbon sources, yeast extract exhibited the highest BR dye decolorization efficiency (88.11%) followed by dextrose (68.32%), sucrose (51.14%), and mannose (13.46), respectively (Figure 2a). Similarly, among the nitrogen sources, ammonium chloride exhibited the highest decolorization efficiency (91.32%) followed by peptone (87.14%), urea (80.24%), and ammonium nitrate (69.54%), respectively (Figure 2b).

FIGURE 2.

Optimization of BR dye decolorization by B. subtilis SC 9 strain. (a) effect of carbon sources; (b) effect of nitrogen sources; (c) effect of pH; (d) effect of temperature. Data represented are average of seven replicates. Data analysis was carried out by one‐way ANOVA using DMRT and treatment means (p < 0.05) were compared. The lowercase letters above the bars denote the significance according to Duncan's Multiple Range Test (DMRT) (p ≤ 0.05).

The effect of pH on the aerobic decolorization of BR dye was also assessed across a pH range of 4–10 (4, 5, 6, 7, 8, 9, and 10) (Figure 2c). The results indicated that the neutral pH favored higher BR dye decolorization efficiency. The highest dye decolorization was observed at pH 7 (92.24%) followed by acidic pH 6 (87.36%), pH 5 (79.54%), and pH 4 (75.62%), respectively. In contrast, alkaline pH lowered the BR dye decolorization rate ranging from 67.33% to 71.22% in the presence of B. subtilis SC 9 (Figure 2c). In terms of temperature, both 37°C (93.10%) and 28°C (84.32%) supported the highest BR dye decolorization by B. subtilis SC 9; whereas, at 30°C BR dye decolorization efficiency was meager and no decolorization was observed at 4°C due to the absence of bacterial growth (Figure 2d). The lower decolorization observed at 30°C may reflect a nonlinear temperature response of the strain under the specific experimental conditions. Although temperature influences bacterial growth and enzyme activity, dye decolorization is also affected by substrate availability, cellular metabolic state, and the expression of dye‐transforming enzymes. Supplementation with yeast extract and ammonium chloride at 37°C having pH 7 significantly enhanced the decolorization efficiency to 88.5%, indicating that external nutrient sources stimulated bacterial growth and dye‐degrading metabolic activity.

2.3. BR Dye Decolorization Analysis by FTIR and GC‐MS Analysis

The FTIR spectrum of the coralene BR dye showed an intense peak at 3356.92 cm⁻¹ for N─H stretching indicating the presence of a primary amide. The peak at 1640.85 cm⁻¹ indicated C═C stretching that is, aliphatic alkene functional group. The presence of secondary amines and amides due to N─H bending and methyl groups due to C─H bending were confirmed by sharp peaks at 1552.18 and 1361.77 cm⁻¹. The peaks at 1337.12, 1156.80, and 974.49 cm⁻¹ confirms C─N stretching, S═O stretching, and C─H bending, respectively, indicating the presence of aromatic primary amines; sulfonate esters sulfonyl chlorides and trans‐alkene functional groups (Figure 3a). On contrast, the BR dye decolorization by B. subtilis SC 9 resulted in the production of some metabolites, which showed peaks at 3443.43 cm⁻¹ for the presence of N─H stretching as primary amines or primary and secondary amides, 3373.82 cm⁻¹ for O─H stretching as alcohols, phenols, 3116.06, 3102.57, and 3077.87 cm⁻¹ for C─H stretching as alkene, aromatic, vinyl, epoxide, and halogen functional groups (Figure 3b). The peak at 2920.76 cm⁻¹ indicated C─H stretching as methyl ketone, 2856.10 cm⁻¹ for C─H stretching as alkyl, 1733.33 cm⁻¹ for α β‐unsaturated and aryl anhydride, 1641.63 cm⁻¹ for C═O stretching as tertiary amide functional groups (Figure 3b). Similarly, the medium strong peak of the metabolites at 1456.19 cm⁻¹ for C─H bending indicated as methylene, 1348.76 cm⁻¹ for C─O─H bending as primary and secondary alcohol, and 1099.58 and 1053.94 cm⁻¹ for C─O stretching as secondary alcohol and primary alcohol. The peaks obtained at 858.41 and 811.67 cm⁻¹ for C─H bending identified as substituted benzene, and a weak bond at 608.94 cm⁻¹ for C─S stretching as thiol and sulfoxide functional groups (Figure 3b). The changes observed in the functional groups of the control and treated BR dye samples supported the decolorization of BR dye by decolorization due to inoculation with B. subtilis SC 9.

FIGURE 3.

FTIR and GC‐MS chromatogram of BR dye before and after treatment with B. subtilis SC 9. (a) FTIR spectrum of BR dye before treatment; (b) FTIR spectrum depicting changes in the BR dye functional groups after bacterial treatment of B. subtilis SC 9; (c) GC‐MS chromatogram of BR dye before treatment; (d) GC‐MS chromatogram indicating changes in the BR dye profile after bacterial treatment of B. subtilis SC 9.

GC‐MS analysis of the BR dye before and after treatment with B. subtilis SC 9 revealed a significant alteration in its chemical composition (Figure 3c,d). The methanolic extract of the untreated dye contained eight distinct compounds, while nine compounds were detected after decolorization of the BR dye by B. subtilis SC 9 (Table 1). Compounds such as 2,2‐dimethoxybutane, butanoic acid, styrene, and butanoic acid, 3‐methyl‐ are found in both untreated and treated BR dye samples. However, compounds such as propanoic acid, 4‐methoxy‐6‐methyl‐3‐(trimethylsilyl) me, butanoic acid, 2‐methyl‐, and glycerin only found in the original untreated BR dye indicating that these compounds might be responsible for its coloration and potential toxic effects (Table 1). In contrast, some of the compounds that were not identified in the BR dye (untreated) but were present in the B. subtilis SC 9 treated BR dye such as toluene, 2‐propanol, 1,1'‐oxybis‐, silane, ethenylmethoxydimethyl‐, isobenzofuran, 1,3‐dihydro‐1,3‐dimethoxy‐, pyrazine, 2,5‐dimethyl‐, and phenol significantly altered the chemical structure of the BR dye indicating the effective decolorization and detoxification by B. subtilis SC 9 (Table 1).

TABLE 1.

Lists of metabolites detected by GC‐MS analysis before and after decolorization of BR dye by B. subtilis strain SC 9.

Before BR dye decolorization			After BR dye decolorization
Peak no	Retention time (min)	Compound name	Peak no	Retention time (min)	Compound name
1	3.209	Propanoic acid	1	4.680	2,2‐Dimethoxybutane
2	4.672	2,2‐Dimethoxybutane	2	4.835	Toluene
3	5.292	Butanoic acid	3	6.713	Butanoic acid, 3‐methyl‐
4	6.707	Butanoic acid, 3‐methyl‐	4	6.838	2‐Propanol, 1,1'‐oxybis‐
5	6.833	4‐Methoxy‐6‐methyl‐3‐(trimethylsilyl) me	5	6.984	Silane, ethenylmethoxydimethyl‐
6	6.909	Butanoic acid, 2‐methyl‐	6	7.153	Isobenzofuran, 1,3‐dihydro‐1,3‐dimethoxy‐
7	7.862	Styrene	7	7.862	Styrene
8	9.572	Glycerin	8	8.377	Pyrazine, 2,5‐dimethyl‐
			9	9.681	Phenol

2.4. Bacillus subtilis SC 9 Encapsulation for BR Dye Decolorization

The calcium alginate method was used to entrap the whole cells of B. subtilis SC 9, where the average size of the calcium‐alginate encapsulated SC 9 alginate beads was found to be 6–7 mm (Figure 4a). For inoculation purposes, 3 g of encapsulated SC 9 beads, and 1 mL of liquid culture of nonencapsulated SC 9 were used. The viability of the cells of encapsulated B. subtilis SC 9 and nonencapsulated B. subtilis SC 9 were found to be 5.4 × 10⁸ and 5.5 × 10⁸ CFU mL⁻¹.

FIGURE 4.

Effect of calcium‐alginate encapsulation on the bioremediation potential of B. subtilis SC 9 against BR dye. (a) formation of calcium‐alginate encapsulated B. subtilis SC 9 beads; (b) BR dye decolorization by nonencapsulated and encapsulated B. subtilis SC 9; (c) percentage of BR dye decolorization by nonencapsulated and encapsulated B. subtilis SC 9; (d) effect of nonencapsulated and encapsulated B. subtilis SC 9 strain on BR dye phytotoxicity on mung bean (Vigna radiata L.). The lowercase letters above the bars in (c) denote the significance according to Duncan's Multiple Range Test (DMRT) (p ≤ 0.05).

2.5. BR Dye Decolorization by Encapsulated and Nonencapsulated B. subtilis SC 9

The efficiency of BR dye decolorization was significantly higher for encapsulated B. subtilis SC 9 than for the nonencapsulated strain when tested with 100 ppm dye under optimal conditions (yeast extract (5 g L⁻¹); ammonium chloride (4 g L⁻¹); pH 7; incubation temperature at 37°C for 9 days). The optimization process of the encapsulated B. subtilis SC 9 alginate beads degraded or decolorized 94.86% of the BR dye under aerobic conditions, whereas the nonencapsulated SC 9 achieved only 88.5% decolorization of the BR dye under the optimized conditions (Figure 4b,c).

2.6. Phytotoxicity Assessment of Coralene BR Dye

The BR dye showed clear phytotoxicity to mung bean plants, where plants exposed to BR dye without bacterial inoculation exhibited wilting of plants and stunted growth. Compared to the control plants (BR dye alone) and nonencapsulated B. subtilis SC 9 inoculated plants, the encapsulated B. subtilis SC 9 inoculated plants were found to have increased plant growth parameters. The increase in root length was 142.16%, shoot length (160.91%), root wet (152.17%), and dry weight (672%), shoot wet (112.58%), and dry weight (147.61%) compared to the control (BR dye inoculation only) (Table 2). The results indicated that the encapsulated B. subtilis SC 9 had a high potential to minimize the toxicity of the BR dye and also has efficacy to enhance the plant growth (Figure 4d and Table 2).

TABLE 2.

Effect of nonencapsulated and encapsulated B. subtilis strain SC 9 on BR dye phytotoxicity of mung bean (Vigna radiata L.).

Treatment	Root length (cm)	Shoot length (cm)	Wet weight (g)		Dry weight (g)
			Root	Shoot	Root	Shoot
Control (no dye or bacterial inoculum)	14.63 ± 3.33b	20.80 ± 0.10b	2.92 ± 0.15b	5.36 ± 0.22ab	0.73 ± 0.15b	2.83 ± 0.16b
BR dye alone	8.30 ± 1.50c	8.93 ± 0.11c	1.38 ± 0.10c	2.86 ± 0.16b	0.25 ± 0.57c	1.26 ± 0.18c
BR dye + B. subtilis SC 9	17.16 ± 2.02ab	21.00 ± 0.10ab	3.00 ± 0.30ab	5.40 ± 0.21ab	1.28 ± 3.00ab	2.98 ± 0.22ab
BR dye + B. subtilis SC 9 beads	20.10 ± 4.22a	23.30 ± 0.17a	3.48 ± 0.15a	6.08 ± 0.17a	1.93 ± 0.20a	3.12 ± 0.11a
LSD (p ≤ 0.05)	1.88	1.88	1.33	1.88	0.021	0.021

Values denoted are Mean ± S.E. of five seedlings, and different lowercase letters denote significant differences between among treatment means (p ≤ 0.05). Values followed by different lowercase letters (a, b, c) are significantly different, while values sharing atleast one common letter (ab) are not significantly different according to Duncan's Multiple Range Test (DMRT) at p ≤ 0.05. One‐way ANOVA followed by Duncan's multiple range test (DMRT) was used for data analysis and treatment means (p ≤ 0.05) were compared. Differences among treatment means were further evaluated using the least significant difference (LSD) test (p ≤ 0.05).

2.7. Soil Respiration Activity

Both nonencapsulated and encapsulated B. subtilis SC 9 strain led to increased soil microbial activity in BR dye contaminated soil. Soils treated with nonencapsulated B. subtilis SC 9 produced 104.24 µg g⁻¹ of TPF through dehydrogenase activity, whereas those treated with encapsulated B. subtilis SC 9 showed a significantly higher production of TPF (192.37 µg g⁻¹) under BR dye induced stress (Figure 5a). Similarly, fluorescein production through FDA hydrolysis was higher in encapsulated B. subtilis SC 9 inoculated soils (33.71 µg g⁻¹) than in the nonencapsulated B. subtilis SC 9 strain (20.00 µg g⁻¹) (Figure 5b). The alkaline phosphatase activity was also found to be high in the case of encapsulated B. subtilis SC 9 (852.20 µg g⁻¹) compared to nonencapsulated B. subtilis SC 9 (584.67 µg g⁻¹) (Figure 5c). The results showed that encapsulation increased the growth of mung bean plants, thereby minimizing the BR dye associated phytotoxicity, and enhanced soil microbial activity (Figure 5).

FIGURE 5.

Effect of calcium‐alginate encapsulated and nonencapsulated B. subtilis SC 9 on soil respiration activity. (a) dehydrogenase assay; (b) FDA hydrolysis assay; (c) alkaline phosphatase assay. Data represented are means of seven replicates. Data analysis was carried out by one‐way ANOVA using DMRT and treatment means (p < 0.05) were compared. The lowercase letters above the bars denote the significance according to Duncan's Multiple Range Test (DMRT) (p ≤ 0.05).

2.8. Siderophore Estimation From BR Dye Stressed Soils

The siderophore production ability of encapsulated and nonencapsulated B. subtilis SC 9 was significantly increased in BR dye amended soils (p ≤ 0.05). The percentage of siderophore production by encapsulated B. subtilis SC 9 was 331.53% compared to nonencapsulated B. subtilis SC 9 (206.97%), which was remarkably higher than the uninoculated negative control (without BR dye; only seeds) (3.61%); and positive control (seeds + BR dye) (20.58%) (Figure 6). This higher production of siderophore suggests that the inoculated encapsulated and nonencapsulated B. subtilis SC 9 scavenged Fe and used this Fe to generate ROS, which might have attacked organic molecules such as dyes; therefore, resulting in the decolorization of BR dye.

FIGURE 6.

Effect of nonencapsulated and encapsulated B. subtilis SC 9 on siderophore production under BR dye (100 ppm) stressed soils. Values denoted are mean ± S.E. (n = 7). Different lowercase letters above the bars denote the significant differences among treatments (p ≤ 0.05).

3. Discussion and Conclusion

3.1. Discussion

Microorganisms utilize complex xenobiotic compounds found in the synthetic dyes as a substrate and play an essential role in breaking them into simpler and less harmful metabolites [23]. Bacterial oxidoreductive enzymes play pivotal roles in decolorization. In this study, bacterial isolates possessing siderophore production ability and other PGP traits were evaluated for their dye decolorization ability. Siderophores play a key role in dye decolorization as the SPB scavenge Fe and use it to generate ROS to attack organic molecules such as dyes that leads to the decolorization of dyes. Moreover, they can chelate metals including those present in dyes, making them soluble so that they can be easily degraded [24].

Among all siderophore producers tested in this study, strain B. subtilis SC 9 exhibited the highest aerobic decolorization efficiency in the presence of 100 ppm BR dye. This strain emerged as the most potent isolate for dye decolorization in the current study, which is consistent with the previous findings of Ikram et al. [25], who reported B. subtilis as an efficient bacterial strain for the reclamation of water loaded with textile azo dye orange II. Abd EI et al. [26] also reported B. subtilis as the most active bacterial strain for its highest bio‐decolorization capacity (71.8% to 100%) of eight azo dyes. B. subtilis not only enhances plant growth through phytohormones, siderophores, ACC deaminase, and phosphate solubilization but also contributes to pollutant degradation by decolorization such as dye remediation. It has been reported that PGPB helps in pollutant breakdown either directly, via enzymatic transformation, or indirectly, by enhancing the decolorization efficiency of other microbes [27, 28].

In the present study, yeast extract and ammonium chloride were used as co‐substrates to enhance the decolorization efficiency of the BR dye by B. subtilis SC 9. The superior performance of yeast extract compared with the tested carbon sources may be attributed to its complex composition of amino acids, peptides, vitamins, minerals, and other growth‐promoting factors. These nutrients can support rapid bacterial growth and provide reducing equivalents required for the enzymatic transformation of azo bonds. Previous work conducted by Imran et al. [29] has also indicated that yeast extract can stimulate azo‐dye decolorization through enhanced azoreductase‐associated activity, providing a mechanistic explanation for the higher decolorization observed in the present study. Ammonium chloride, an inorganic nitrogen source, facilitates protein and enzymes synthesis, which are crucial for the breakdown of complex dye molecules. Previous studies also support the present findings. Anwar et al. [30] and Mahmood et al. [31] demonstrated the yeast extract as an effective carbon source for enhancing microbial activity and dye decolorization. Similarly, Tripathi et al. [32] reported that maltose and ammonium chloride enhanced the generation of secondary electron donors during microbial respiration, which has led to the reduction of azo bonds in dye molecules. These findings support the use of carbon and nitrogen co‐substrates to improve the bioremediation potential of dye‐degrading bacteria. Although yeast extract produced the highest decolorization in the present optimization experiment, its relatively high cost may limit large‐scale application. Agricultural residues such as molasses, rice bran hydrolysate, wheat bran hydrolysate, fruit‐processing residues, or other locally available organic wastes may provide lower‐cost carbon and nutrient sources. Future studies should evaluate these substrates as alternatives to yeast extract.

In terms of pH and temperature, the highest BR dye decolorization was achieved at 37°C with a neutral pH (pH 7) over a 9‐day incubation. Acidic pH diminished the decolorization efficiency compared to alkaline pH, possibly because of reduced enzyme activity or bacterial viability. The present results are in agreement with those of Wang et al. [33] and Veismoradi et al. [34], who highlighted the importance of optimizing the environmental conditions for microbial dye decolorization. Although maximum decolorization was obtained at pH 7, B. subtilis SC9 maintained measurable decolorization activity under alkaline conditions (pH 8–10), indicating a degree of tolerance to alkaline environments. Since textile wastewater is generally alkaline, practical application may involve partial pH adjustment before biological treatment or gradual adaptation of the bacterial culture to alkaline conditions. Future studies should investigate long‐term adaptation and continuous bioreactor performance under alkaline textile wastewater conditions

The enhanced decolorization efficiency observed in the present study can be attributed to the improved metabolic activity of B. subtilis SC9 under optimized nutritional conditions. Appropriate nutrient supplementation promotes bacterial growth and stimulates the production and activity of dye‐degrading enzymes, particularly azoreductases and other oxidoreductases, thereby accelerating the biodegradation of azo dyes into less toxic intermediates. Moreover, siderophore production may further facilitate this process by improving iron acquisition, which is essential for the activity of several iron‐dependent oxidoreductases involved in microbial metabolism and dye transformation. Enhanced iron availability supports bacterial physiological functions and enzymatic efficiency, ultimately contributing to more effective dye degradation and detoxification. These observations are consistent with previous reports conducted by Imran et al. [35], and Abbas et al. [36] demonstrating that optimized nutritional conditions and enhanced azoreductase activity significantly improves microbial azo dye degradation and detoxification processes. The One‐Factor‐at‐a‐Time (OFAT) approach was employed as an initial optimization strategy to determine the individual influence of carbon source, nitrogen source, pH, and temperature on BR dye decolorization. Although statistical optimization methods such as Response Surface Methodology (RSM) provide better interaction analysis, OFAT remains useful during preliminary screening because it identifies critical variables and establishes baseline operating conditions prior to advanced optimization [37].

Furthermore, FTIR analysis confirmed the structural transformation of the BR dye by B. subtilis SC 9 treatment indicating the cleavage of the azo bond and alteration of the chromophoric structure of the dyes. Similar findings have been reported by Lade et al. [38] and Mahajan et al. [39]. The present findings are consistent with recent reports conducted by Kumar et al. [40], demonstrating the efficient biodegradation of azo dyes by bacterial strains, where spectroscopic analyses (FTIR and UV‐Visible spectroscopy) confirmed structural transformation and detoxification of dye molecules. GC‐MS analysis further supported the decolorization results by identifying distinct metabolites with altered retention times and mass‐to‐charge ratios [41, 42]. Compounds such as toluene, styrene, and phenol were reported as intermediates in the degradation of azo and other synthetic dyes resulting from the cleavage of azo bond due to reductive breakdown of the aromatic backbone [8]. Phenol was recognized as hydroxylated aromatic intermediates as a result of microbial oxidation of dye‐derived benzene rings [43]. Similarly, the presence of isobenzofuran, 1,3‐dihydro‐1,3‐dimethoxy‐ and 2,5‐dimethylpyrazine suggests transformation of the dye's complex aromatic core into oxygenated heterocyclic metabolites, consistent with reported pathways of dye mineralization [44]. Thus, the presence of these specific intermediates confirms the ability of B. subtilis strain SC 9 to degrade BR dye to simpler aliphatic acids through sequential aromatic cleavage, and hydroxylation.

Bacillus subtilis SC 9 was encapsulated in calcium alginate beads resulting in significant higher aerobic decolorization rate (94.86%) compared to free cells (84.67%). Encapsulation offers advantages by maintaining enzyme integrity and shielding cells from toxic stresses such as dye [34]. Organic compounds get adsorbed onto the carrier material surface first and then penetrates through pores, where microbes slowly release extracellular enzymes in order to pre‐hydrolyze the organic xenobiotics which are then transported through the cellular membrane for oxidation. Encapsulation provides a stable microenvironment, facilitates substrate diffusion, and is considered a mild, nontoxic method that is suitable for microbial entrapment [45]. The superior performance of encapsulated cells may be attributed to the protective microenvironment created by calcium‐alginate beads, which minimizes direct exposure of bacterial cells to toxic dye molecules while maintaining higher metabolic activity. The porous structure of alginate permits diffusion of nutrients and dye molecules while preventing excessive cell loss. Encapsulation also improves operational stability, enhances resistance to environmental stress, and increases the potential for repeated use in wastewater treatment systems [46]. Phytotoxicity studies on mung bean plants revealed that BR dye had detrimental effects on root and shoot growth in control plants where no bacteria were inoculated, whereas treatments with both encapsulated and free B. subtilis SC 9 cultures significantly improved plant physiological parameters. This suggested that the degraded metabolites were less toxic than the parent dye. Similar findings have been reported by Singh et al. [46] and Pandey et al. [47]. The higher decolorization efficiency observed with calcium‐alginate‐encapsulated B. subtilis SC9 (94.86%) compared with free cells suggests that encapsulation effectively maintained bacterial viability and metabolic activity throughout the incubation period. Although quantitative measurements of immobilization efficiency and cell leakage were not performed, successful recovery of viable colonies from the alginate beads confirmed that bacterial cells remained viable after encapsulation. Future studies should quantify immobilization efficiency, cell leakage, and bead stability during repeated operational cycles to further evaluate the long‐term applicability of the immobilized system.

Furthermore, the presence of BR dye alone did not significantly alter the soil respiratory activity. However, soils inoculated with B. subtilis SC 9 showed increased soil respiratory enzyme activities such as dehydrogenase, alkaline phosphatase, and FDA indicating increased microbial activity in the soil. This effect was more pronounced in soils treated with encapsulated bacteria, as reported by Omar et al. [48], Sritongon et al. [49], Chandwani et al. [15], Chukwuneme et al. [18], Taha and Gouda [16], and Trivedi and Sa [50].

3.2. Conclusion

The present study demonstrated that the siderophore‐producing B. subtilis SC9 effectively decolorized BR dye exhibiting 84.67% decolorization without optimized conditions, 88.5% decolorization under optimized conditions, and 94.86% decolorization after calcium alginate encapsulation, confirming the superiority of immobilized cells over free bacterial cells. The production of siderophores by B. subtilis SC9 likely contributed to the biodegradation process by improving iron acquisition, stimulating bacterial metabolic activity, and supporting the activity of dye‐degrading enzymes under nutrient‐limited conditions. FTIR and GC‐MS analyses confirmed the transformation of BR dye into less complex intermediates, indicating efficient biodegradation rather than simple adsorption. Greenhouse experiments further revealed that encapsulated B. subtilis SC9 significantly alleviated BR dye‐induced phytotoxicity by improving the growth attributes of mung bean (V. radiata L.) (root length and shoot length by 142.17% and 160.92%, respectively) and enhancing soil enzyme activities, including dehydrogenase (192.37 µg TPF g^{− 1} soil), fluorescein diacetate (FDA) hydrolysis (33.71 µg fluorescein g^{− 1} soil), and alkaline phosphatase (852.20 µg pNP g^{− 1} soil) activities compared with the untreated dye treatment. These findings demonstrate that bacterial encapsulation not only improves microbial survival and functional stability but also enhances the beneficial effects of siderophore‐producing bacteria under dye‐stressed conditions.

Overall, this study highlights the synergistic role of siderophore production and calcium alginate encapsulation in promoting efficient dye biodegradation, restoring soil biological activity, and supporting sustainable plant growth in dye‐contaminated environments. The developed encapsulated bacterial formulation represents an eco‐friendly and cost‐effective strategy for the remediation of textile dye‐polluted soils and wastewater.

Future research should focus on validating the technology under field conditions, elucidating the molecular mechanisms underlying siderophore‐mediated dye biodegradation and plant growth promotion, determine the activities of azoreductase, laccase, lignin peroxidase, manganese peroxidase, and related oxidoreductases to elucidate the molecular mechanism of dye degradation, evaluating the long‐term stability and reusability of encapsulated bacterial formulations, and exploring their applicability for the remediation of a wider range of industrial dyes and contaminated environments. Also, future studies should investigate low‐cost agricultural residues such as molasses, rice bran, wheat bran, sugarcane bagasse hydrolysate, corn steep liquor, or fruit‐processing wastes as alternative carbon sources of yeast to improve the economic feasibility of the process. The decolorization efficiency of encapsulated B. subtilis SC9 at higher dye concentrations (150–250 mg L^{− 1} or higher) to better simulate textile wastewater conditions also need to be explored in future.

4. Materials and Methods

4.1. Collection of BR Dye and Bacterial Cultures

The previously isolated and identified ten bacterial cultures (CWN 10, SKTS 11, KSBTS 12, KBN 12, KBN 15, SC 9, SC 7, CWTS 5, CWTS 10, and IPTS7) possessing siderophore production ability were revived on nutrient agar medium and then screened on Chrome Azurol S agar plates to reconfirm the siderophore production ability of the bacterial isolates [51]. The ten SPB also possessed phosphate solubilization, indole acetic acid (IAA)‐like substance and 1‐aminocyclopropane‐1‐carboxylic acid (ACC) deaminase production ability from the previous study. Coralene BR dye was purchased from the textile industry of Ankleshwar, Gujarat (India) (Product code: T034480126; Coralene Brill, Red F3BS; Ref. No. 0001236788; manufacturer: Colortex dye stuff company, Surat, India).

4.2. Screening of SPB for BR Dye Decolorization

Ten SPB were preliminarily screened out for dye decolorization efficiency qualitatively on nutrient agar medium (Peptone, 5.0 g L^{− 1}; beef extract, 1.5 g L^{− 1}; yeast extract, 1.5 g L^{− 1}; sodium chloride, 5.0 g L^{− 1}; agar, 15.0 g L^{− 1}; pH 7.4 ± 0.2 (25°C)) containing 100 ppm of coralene BR dye. The four isolates that showed a zone of BR dye decolorization that is, CWTS 10, IPTS 7, KBN 12, and SC 9 were further quantitatively screened for BR dye decolorization [52]. Nutrient broth (100 mL) (Peptone, 5.0 g L^{− 1}; beef extract, 1.5 g L^{− 1}; yeast extract, 1.5 g L^{− 1}; sodium chloride, 5.0 g L^{− 1}; pH 7.4 ± 0.2 (25°C)) was prepared in a 250 mL Erlenmeyer flask and sterilized at 121°C and 15 lbs pressure for 15 min. Thereafter, 100 ppm (0.1%) BR dye solution was sterilized separately by filtration through a sterile membrane filter and aseptically added to the cooled sterile medium, where 5.6 × 10⁸ CFU mL⁻¹ bacterial cells were suspended in the medium containing BR dye and incubated at 28°C (Incubator manufacturer: Thermo Fisher Scientific, USA, Model 51028063) for 9 days. Dye medium without bacterial inoculation serves as a control. Samples (10 mL) were withdrawn every 3 days for 9 days and centrifuged at 10 000 rpm for 10 min at room temperature (RT) (Cooling centrifuge manufacturer: Centurion Scientific Ltd., UK, Model K242R). The absorbance of the supernatant was measured at the maximum wavelength (630 nm) using a spectrophotometer (Manufacturer: Shimadzu Corporation, Kyoto, Japan, Model UV‐1800). The experiment was conducted in triplicate and repeated twice to ensure accuracy of the results. The percentage (%) of dye decolorization was calculated as follows

$$\mathrm{Dye}\mathrm{decolorization}\left(\%\right)=\frac{\mathrm{Initial}\mathrm{OD}-\mathrm{Final}\mathrm{OD}}{\mathrm{Initial}\mathrm{OD}}\times 100$$

4.3. Optimization of BR Dye Decolorization by B. subtilis SC 9

The strain B. subtilis SC 9 (GenBank Accession no. ON377344), which was found to be efficient in BR dye decolorization both qualitatively and quantitatively was further optimized for efficient decolorization. The parameters such as carbon and nitrogen source, pH and temperature were modified to increase the efficiency BR dye decolorization. To optimize the effects of carbon and nitrogen sources, the nutrient broth was supplemented with 5 g L⁻¹ of carbon sources such as mannose, sucrose, yeast extract, and dextrose, and 4 g L⁻¹ of nitrogen sources such as ammonium nitrate, peptone, ammonium chloride, and urea along with 100 mg L⁻¹ of BR dye. The reaction mixtures were then incubated for 9 days at 37°C to assess the efficacy of the isolate for BR dye decolorization. Similarly, to optimize the pH conditions, a range of 4–10 pH was considered, in which the broth amended with 100 mg L⁻¹ of BR dye was incubated at 37°C for 9 days. The effects of various temperatures such as 4°C, 28°C, and 37°C were also assessed in the BR dye decolorization assay. Dye medium without bacterial inoculum serves as a control. All optimized parameters were assessed in seven replicates aerobically under static conditions. Static incubation was selected to evaluate decolorization under conditions that minimize mechanical energy input and permit direct comparison of bacterial activity under non‐agitated conditions. This approach also provides a baseline for evaluating the potential of the system for low‐energy treatment applications. The decolorization percentage of the BR dye was measured as mentioned above [53].

4.4. Assessment of BR Dye Decolorization Efficiency via FTIR and GC‐MS Analysis

The change in the chemical composition of the BR dye was determined via FTIR and GC‐MS analyses [15]. To assess the effect of SPB on BR dye decolorization, B. subtilis SC 9 was inoculated into a nutrient broth containing 100 ppm of BR dye and then incubated at 37°C for 9 days under aerobic static conditions. BR dye without inoculation of the bacterial culture served as a control. After 9 days of incubation, both the control broth and the bacterial culture treated broth were centrifuged at 10 000 rpm for 10 min to collect the supernatant. The collected supernatant was further filtered through 0.45 µm filter to obtain a clear filtrate. The clarified filtrate was then mixed with an equal volume of methanol and the organic phase was allowed to evaporate. The derived extract was then analyzed using FTIR and GC‐MS. For FTIR spectroscopy, translucent sample discs were prepared by encapsulating 3 mg of the dried dye‐degrading extract in 30 mg of KBr pellets. The prepared extract was then loaded into an FTIR spectroscope with a scan range of 400–4000 and 4 cm⁻¹ resolution. The functional groups present in the dye‐degrading extracts were determined based on the obtained IR spectrum. The dye‐degrading extract was further subjected to GC‐MS analysis as per the standard protocol of Jung and Jung [54]. The analysis was carried out at the Sophisticated Analytical Instrument Facility (SAIF), IIT Bombay, Mumbai, India. The GC‐MS system was equipped with DB‐5MS capillary column, where 1 µL sample was injected with an injector temperature of 250°C using helium as the carrier gas at a constant flow rate of 1.0 mL min^{− 1}. Mass spectra were recorded with electron impact ionization (70 eV) over a scan range of m/z 35–600. The resulting mass spectra and chromatograms were compared with the NIST library database for the identification of the compounds.

4.5. Encapsulation of B. subtilis SC 9

The encapsulation of B. subtilis SC 9 was carried out through an ionotropic gelation. For this, 2 g of sodium alginate was dissolved in 100 mL of sterile distilled water and heated gently to dissolve the sodium alginate clumps. An overnight grown culture of B. subtilis SC 9 was centrifuged at 10 000 rpm for 10 min and the resulting pellet was resuspended in phosphate‐buffered saline (PBS). The bacterial suspension was then mixed with sodium alginate solution in a 1:1 ratio. A 2% (w/v) calcium chloride (CaCl₂) solution was prepared and the alginate‐bacteria mixture (approximately 5.4 × 10⁸ CFU mL⁻¹) was extruded dropwise into 200 mL of CaCl₂ solution with the help of a syringe, forming alginate beads of 5–6 mm diameter. After 20 min, the alginate beads were collected by filtration and stored in 0.5% CaCl₂ solution at 4°C for 10–12 days. Prior to use, the beads were air‐dried for 2 h and rinsed with sterile distilled water. The viability and colony‐forming units (CFU) of the encapsulated bacteria were assessed on nutrient agar following the method described by Steffan et al. [45, 55].

4.6. BR Dye Decolorization by Encapsulated and Nonencapsulated B. subtilis SC 9

The BR dye decolorization ability of encapsulated and nonencapsulated cultures of B. subtilis SC 9 was conducted as described above. In the experimental setup, 3 g of encapsulated cells (5.4 × 10⁸ CFU mL⁻¹) and 1 mL of nonencapsulated bacterial culture (5.5 × 10⁸ CFU mL⁻¹) were introduced into separate flasks containing nutrient broth with 100 ppm BR dye. The flasks were then incubated under aerobic conditions at 37°C for a period of 9 days to monitor the BR dye removal potential of both nonencapsulated and encapsulated bacterial cultures of B. subtilis SC 9 [56]. The dye medium without bacterial inoculum serves as a blank or a control.

4.7. Phytotoxicity Assessment

The experiment aimed to assess the impact of both encapsulated and nonencapsulated B. subtilis strain SC 9 on V. radiata in the presence of 100 ppm BR dye. Phytotoxicity studies were conducted to assess the potential toxicity of the BR dye and its decolorized products on plant growth. Vigna radiata L. (mung bean) var. Meha seeds were procured from a local market in Bardoli (Gujarat, India). The physicochemical parameters of the sandy soil used in the greenhouse experiment were as follows: pH 7.3, electrical conductivity 0.188 mLmho cm⁻¹, available phosphorus 136.34 mg kg⁻¹, nitrogen 124.31 mg kg⁻¹, potassium 396.99 mg kg⁻¹, and sulfur 1.95 mg kg⁻¹. The pots were filled by mixing 2 kg of sandy soil with 100 ppm BR dye. For the phytotoxicity experiment, BR dye was directly incorporated into the soil at 100 ppm. The bacterial treatments consisted of either free‐cell culture or calcium‐alginate‐encapsulated cells directly mixed into the dye‐amended soil. Ten mung bean seeds were sown per pot. Bacterial treatments (encapsulated or nonencapsulated) were uniformly mixed into the soil to ensure even distribution. The treatments included (i) negative control (no BR dye or bacteria), (ii) positive control (BR dye only), (iii) BR dye with nonencapsulated bacteria (5.5 × 10⁸ CFU mL⁻¹), and (iv) BR dye with encapsulated bacteria (3 g containing 5.4 × 10⁸ CFU mL⁻¹). A completely randomized design was adopted with seven replicates per treatment under greenhouse conditions under natural daylight with an average daytime temperature of 28°C ± 2°C and 65% relative humidity. The pots were irrigated with sterile water every 2 days interval in order to maintain the optimal soil moisture without causing waterlogging. After 30 days, the plants were harvested, and root length, shoot length, and both wet and dry biomass were measured. Biomass values were expressed as g plant^{− 1} [57].

4.8. Estimation of Soil Respiration Enzymes

To evaluate the effects of BR dye toxicity on soil microbial activity, soil respiration enzymes such as FDA hydrolysis, alkaline phosphatase, and dehydrogenase were analyzed. FDA hydrolysis was assessed following the method of Adam and Duncan [58], in which FDA was enzymatically cleaved to release fluorescein. Alkaline phosphatase was determined using the method described by Otto et al. [59], where para‐nitrophenyl phosphate (pNPP) was used as a substrate to quantify the release of para‐nitrophenol (PNP). The dehydrogenase activity was measured according to the method of Casida et al. [60] using 2‐3‐5‐triphenyl tetrazolium chloride (TTC) as a substrate, which was then reduced to triphenyl formazan (TPF) as an indicator of microbial oxidative activity. The untreated soil sample (no BR dye and bacteria), and soil with BR dye only serves as the control for this experiment. All enzymatic analyses were conducted in triplicate to ensure reproducibility.

4.9. Estimation of Siderophore from BR Dye Stressed Soils

Siderophore production in dye stressed soils was estimated in triplicate using the modified Chrome Azurol S (CAS)‐Fe agar method, as described in our study [61]. In this assay, the pH of the Chrome Azurol S reagent was adjusted to a neutral range of 6.8–7.2 to ensure the optimal assay performance because siderophore activity is pH sensitive. Maintaining a neutral pH facilitates the accurate detection of color changes and minimizes the risk of false‐negative results. The untreated soil sample (no BR dye and bacteria), and soil with BR dye only serves as the control for this experiment.

4.10. Statistical Analysis

Statistical analysis was performed using the IBM SPSS software version 23.0. Data were subjected to one‐way analysis of variance (ANOVA) followed by Duncan's multiple range test (DMRT) to determine the significant differences among treatment means. Statistical significance was set at p < 0.05. Results are expressed as the mean ± standard error (SE).

Author Contributions

N.A. designed the study. S.C., M.R., and A.G. performed the experiments and analyzed the data. S.C. and N.A. wrote the manuscript.

Funding

The authors have nothing to report.

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

All data generated or analyzed during this study are included in this published article