Abstract
The present study evaluated bioaugmented floating treatment wetlands (FTWs) for the effective remediation of textile waste enriched with the azo dyes Synozol Red K3 BS (SR), Synozol Yellow K3 RS, and Synozol Ultra Black DR (SB). Six strains of bacteria (SZ1-SZ6) were isolated, characterized and tested for the ability to decolorize dyes. Of these, the SZ1 strain, Bacillus sp., was observed to have a maximum removal rate of 92% for SR, 89.96% for SY and 54% for SB dyes in shake flask assays. The performance of SZ1 was further optimized under different physicochemical conditions, pH (1, 3, 5, 7, 9, 11, and 13), incubation temperature (30–40°C), incubation days (1, 3, 5, and 7), and inoculum size (1, 2, 3, and 4%). The results indicated that optimum conditions for maximum decolorization were at pH 7, temperature 37°C and 2% inoculum size. SZ1 was then optimized and further introduced in FTWs vegetated with Typha domingensis. Bioaugmented FTWs (T. domingensis + Bacillus sp.) achieved significantly more dye removal up to 95% compared with FTWs with only plants or bacteria. Moreover, growth parameters (root and shoot length) were improved in bioaugmented systems indicating reduced toxicity. The results demonstrated that Bacillus Sp. and T. domingensis were complementary effective in the performance of FTWs to treat the dye-contaminated water, which provided an environmentally friendly FTW-based remediation solution as compared to the conventional one.
Graphical Abstract
Introduction
The textile industry is a dynamic component of the economy of most countries, including Pakistan, and is an important source of employment, industrial output, and export earnings (; ). Although the industry has economic benefits, it poses substantial environmental challenges as a consequence of significant amounts of untreated effluents discharged, including synthetic dyes, mostly azo dyes (). The annual production of synthetic dyes is expected to be 700,000 tons, which equals a total of around 280,000 tons of untreated dye compounds entering the surface waters of the world, where they can be found (; ). These dyes lead to decreased light penetration, increased biological oxygen demand (BOD) and chemical oxygen demand (COD), disruption of aquatic photosynthesis, and even the release of toxic or carcinogenic aromatic amines during degradation (; ). The treatment of dyes in industrial effluents by physicochemical methods has been used for many years. The methods used are coagulation, flocculation, advanced oxidation processes, and membrane filtration ().
Such techniques, however, come with some limitations, such as higher operational expenses, energy consumption, and the generation of secondary waste, or sludge, which needs more treatment and disposal (; ). Biological treatments provide a promising, cost-effective, and environmentally friendly energy solution in comparison (; ; ; ). Bioremediation is one of the biological approaches which involves the use of living organisms, such as microorganisms and plants, and has been identified as a cost-effective and sustainable solution (; ). Azo dyes can be biotransformed by the action of microorganisms using enzymatic processes such as manganese peroxidase, lignin peroxidase, azo reductases, and laccases or by biosorption onto their cell walls (; ; ). The removal of dyes by microorganisms is complemented by aquatic plants by the process of rhizodegradation or phytodegradation. The plant roots exude a number of substances that enhance the microbial activity in the rhizosphere and plant peroxidases and laccases may directly transform/degrade dye molecules (; ). Therefore, the synergistic effect of the plant and microorganism is responsible for the FTWs which is a promising natural solution to treat dye polluted water.
FTWs are designed systems that use aquatic plants atop buoyant mats that let the roots of the plants hang into the water to replicate natural wetlands. The roots broaden the surface area available to the microorganisms to colonize, allowing them to form a symbiotic relationship where plants and microorganisms work together to mineralize and detoxify pollutants (; ). Recent studies have shown that the use of dye-decolorizing bacteria (DDs) can greatly enhance the performance of FTWs (; ; ; ). In this context, the present work aims to isolate and select an indigenous bacterial strain capable of decolorizing industrially relevant azo dyes. The selected strain was applied in FTWs vegetated with the native wetland plant Typha domingensis to evaluate enhanced remediation potential. To the best of our knowledge, unlike previous studies that have largely focused on single-model dyes, this study investigates the remediation of multiple commercially relevant azo dyes (Synozol red, yellow, and black), thereby better representing the complexity of textile effluents.
Experimental section
Reactive dyes
Three azo dyes, Synozol Red K3 BS (SR, C.I. Reactive Red 195), Synozol Yellow K3 RS (SY, C.I. Reactive Yellow 145), and Synozol Ultra Black DR (SB, C.I. Reactive Black 5), were purchased from a chemical market in Lahore, Pakistan.
Collection of wastewater and physicochemical examination of industrial effluent
Textile effluent was collected from the industrial area of Kot Lakhpat, Lahore, Pakistan, and stored at 4°C until use. The physical parameters, such as color, pH, and temperature, of the sample were recorded on-site, while BOD, COD, total dissolved solids (TDS), and total suspended solids (TSS) were measured as described by .
Screening and characterization of dye-decolorizing bacteria
Strains of bacteria capable of growing on azo dyes (SB, SY, SR) were isolated by enrichment. 20 mL of textile effluent was added to 80 mL of MSM containing 50 mg/L each of the reactive dyes, and the mixture was incubated at 37°C for 7 days. After incubation, serial dilutions were coated on MSM agar plates supplemented with 50 mg/L of each reactive dye. Plates were left to incubate at 37°C for 72 h. After 72 h, individual bacterial colonies were picked and purified by the sub-culturing technique (). Morphological characterization, followed by Gram staining, was performed to study the characteristics ().
Initial screening of dye-decolorizing bacteria in a shake flask using UV-Vis spectrometry
The cultures were cultured for 7 days after a loopful of each of the six colonies was individually inoculated into MSM supplemented with 50 mg/L of each reactive dye. The biomass and supernatant were then separated by centrifuging the samples for 1 min at 13,000 rpm. The supernatant was used to determine the concentration of azo dyes by measuring absorbance at 600 nm with a spectrophotometer (). The dye decolorization rate (%) was measured using the following formula.
Evaluation of dye tolerance in bacterial strains at varying concentrations of azo dyes
The dye tolerance of six isolated strains was assessed by exposing them to azo dyes at increasing concentrations (50, 100, 150, 200, and 250 mg/L). The survival of bacteria under dye-induced stress conditions was monitored by incubating cultures at 37°C for 96 h. Uninoculated flasks containing the same dye concentrations served as controls.
Identification through 16S rRNA gene sequencing
Using conventional primers 27F and 1492R for PCR amplification of the 16S rRNA gene, the top-performing bacterial strain, SZ1, was found. The NCBI BLAST program1 was used to compare the acquired sequence with sequences in the NCBI GenBank database. The Multiple Alignment Fast Fourier Transform (MAFFT) at EMBL-EBI2 was used to combine and align query and reference sequences. MEGA XII (Molecular Evolutionary Genetics Analysis-XII) was used to create a phylogenetic tree using the obtained alignment and tree files. The tree was visualized and edited using the iTOL web server (Interactive Tree of Life)3 ().
Growth optimization of SZ1 at various physicochemical parameters
The strain SZ1 was examined under varying set of conditions (ranges from 1, 3, 5, 7, 9, 11, and 13), incubation temperature (ranges from 30 to 40°C), incubation days (1, 3, 5, and 7) and inoculum size (1, 2, 3, and 4%) in order to maximize the decolorization potential ().
Development of floating treatment wetlands
Establishment of reactive dye-enriched industrial wastewater
Wastewater containing reactive dye was synthetically prepared at a dye concentration of 100 ppm.
Plant
Typha domingensis (southern cattail) was harvested from Minhala village near the Wahga Border in Lahore. It was selected as it is native and adaptable to diverse environmental conditions, and has been reported to enhance the removal of dyes and other organic pollutants.
Bacterial inoculum preparation
A polystyrene sheet was used to create 36 FTW microcosms in total. Five healthy T. domingensis seedlings could fit in the hole made in the middle of the sheet after it was cut in a circle (Figure 1). This setup was placed on top of the water tanks containing 5 L of uncontaminated water. The plants were allowed to grow under uncontaminated water conditions, which were subsequently substituted with reactive dyes after 15 days. The experiment was run in triplicate. The details are as Control 1 (C): Water contaminated with dye SR; Control 2 (C): Water contaminated with dye SY; Control 3 (C): Water contaminated with dye SB; Treatment (T1): Water contaminated with SR and SZ1; Treatment (T2): Water contaminated with SY and SZ1; Treatment (T3): Water contaminated with SB and SZ1; Treatment (T4): Water contaminated with dye SR and T. domingensis; Treatment (T5): Water contaminated with dye SY and T. domingensis; Treatment (T6): Water contaminated with dye SB and T. domingensis; Treatment (T7): Water contaminated with dye SR, T. domingensis and SZ1; Treatment (T8): Water contaminated with dye SY, T. domingensis and SZ1; Treatment (T9): Water contaminated with dye SB, T. domingensis and SZ1.
FIGURE 1
Determination of plant growth and analysis of residual reactive dye concentrations in water
The shoot and root lengths of T. domingensis were measured after 90 days to assess the effects of reactive dye toxicity and bacterial inoculation on plant growth. The effluent collected after on-site treatment was evaluated for dye degradation (%) using a UV-Visible spectrophotometer, as described previously.
Phytotoxicity assay
Before and after treatment in FTWs, the toxicity of azo dyes to Triticum aestivum seeds was evaluated using a phytotoxicity test. After soaking in 0.1% HgCl2 for 2 min to surface-sterilize each seed, sterile distilled water was used to rinse them all. The seeds (n = 20) were placed in autoclaved Petri plates coated with blotting paper. For 5 days, 2 mL of tap water, dye-contaminated water (100 ppm), and treated water were added to petri plates. After 5 days, seed germination %, shoot, and root length were measured.
Statistical analysis
Analysis of variance (ANOVA) was utilized to evaluate significant differences between treatments after data were processed using SPSS (SPSS Inc., Chicago, IL, United States). Statistical significance was determined using a p-value of < 0.05. A post-hoc Tukey’s HSD test was subsequently conducted at α = 0.05 to assess significant differences.
Results and discussion
Isolation and in vitro dye decolorization assay
The physicochemical characteristics were determined by analyzing the wastewater sample. The wastewater exhibited a pH of 6.8, a COD of 1,171 mg/L, and a BOD of 130 mg/L, indicating a considerable organic pollution load. Six isolated bacteria were obtained from the textile effluent samples and were named SZ1 through SZ6. Only two isolates (SZ1 and SZ6) were found to show visible color zone around their colonies by initial screening, showing some dye-decolorizing capability. These strains were also assessed in the decolorization of these dyes by shake flask assay. ANOVA revealed significant effects of both dye type and bacterial strains on decolorization efficiency (p < 0.05). Among all strains, SZ1 and SZ6 showed 92–84% decolorization of SR, 89.96–80% decolorization of SY, and 54–50% decolorization of SB dyes, respectively (Figure 2). A consistent pattern was observed across decolorization for all isolates, i.e., monoazo (SR and SY) > diazo dyes (SB). Thus, decolorization is significantly linked to both dye complexity and bacterial strain. The capacity of different organisms to degrade dyes varies with the dye type, the organism, and numerous environmental factors, such as temperature and pH (; ). The current findings are in line with research conducted by Emadi et al. and , which found that diazo dyes (AB-113: acid blue 113) decolorized more slowly than mono-azo dyes (MR: methyl red and MO: methyl orange).
FIGURE 2
Molecular identification and phylogenetic analysis
The best-performing strain, SZ1, was identified by nucleotide BLAST (BLASTn) with a cutoff of > 95%. BLAST analysis revealed that the query sequence exhibits 100% similarity with Bacillus thuringensis, Bacillus paramycoides, Bacillus anthracis, and Bacillus albus. Sequences were then extracted from the NCBI GenBank database in FASTA format. A phylogenetic tree was then built using these sequences (Figure 3). Phylogenetic analysis revealed that strain SZ1 clustered with Bacillus genus, however, the 100% sequence identity observed across the four species in the sequenced region excluded species-level discrimination (). Consequently, strain SZ1 was tentatively identified as Bacillus sp. (GenBank accession number: PZ493129). Further molecular techniques, such whole-genome sequencing (WGS) or multilocus sequence typing (MLST), would be necessary for species-level categorization (; ). Congo Red, Reactive Black 5, Reactive Green 19, Reactive Red 120, and Reactive Blue 4 were among the azo dyes that Bacillus subtilis CKCC decolorized. Additionally, under anaerobic circumstances, Bacillus cohnii, Bacillus sp. YZU1, and Bacillus pumilus were reported to decolorize direct dyes (; ). Thus, Bacillus spp. are widely prevalent in dye-contaminated environments (; ) because of their ability to tolerate high concentrations of reactive azo dyes, diverse enzymatic activity (azoreductase and ligninolytic enzymes such as laccase, manganese peroxidase and lignin peroxidase), and adaptability, which makes them key members of microbial communities in textile effluent and wastewater treatment systems.
FIGURE 3
Influence of dye concentration on azo dye decolorization
The maximum decolorization was found at 50 mg/L and 100 mg/L dye concentration for SR dye (∼90–92%), SY dye (∼85–89%) and SB dye (∼51–54%). Further reductions were noted at 150 mg/L (SR 70%, SY 60%, SB 46%), 200 mg/L (SR 60%, SY 40%, SB 30%), and 250 mg/L (SR 30%, SY 20%, SB 10%) (Figure 4). The dye concentration significantly affected the decolorization efficiency (p < 0.05). The highest decolorization was recorded at 50 mg/L, followed by 100, 150, 200, and 250 mg/L, indicating a progressive decrease in decolorization with increasing dye concentration. The reduction in the decolorization percentage might be due to a higher concentration of dye molecules blocking enzyme active sites or to dye toxicity. Similar studies have also reported that lower dye concentrations, such as 100 mg/L, are optimal for decolorization. The literature indicates that lower dye concentrations reduce the toxic load on bacterial cells, thereby ensuring optimal growth and activity (). In contrast, higher dye concentrations limit decolorization by inhibiting microbial metabolism and enzyme activity through toxic effects.
FIGURE 4
Optimization of dye degradation by various physicochemical parameters
The results indicate that maximum dye decolorization occurred at pH 7, a 2% inoculum size, a 7-day incubation period, and 37°C (Figure 5). At pH 7, the decolorization percentages were approximately 92% for SY dye, 89% for SR dye, and 54% for SB dye (Figure 5a). With a 2% inoculum size, red and yellow dyes achieved around 88–85%, while black dye achieved a 50% decolorization. With a 7-day incubation period, yellow and red achieved maximum decolorization (∼90 and 89%, respectively), while black dye reached a maximum of 49%. At 37°C, red and yellow dyes achieved removal rates of around 88 and 82%, while black remained at about 49% (Figure 5b). Dye decolorization was significantly lower at extreme pH values, at very low or high inoculum sizes (1 and 4%), and at short incubation periods (Figure 5c), indicating that dye removal depends on various optimal physicochemical and biological factors. This is probably due to the fact that the values of pH 7 are optimum for Bacillus sp., and any extremes of pH can inactivate several enzymes that break down dyes, including azoreductases, peroxidases, and laccases (). The microbial biomass is enough to decolorize azo dyes but not compete for nutrients in the medium if the inoculum size is kept at 2% (Figure 5d). The 7-day incubation period allows bacteria to grow, express enzymes and completely break down the dye molecules, especially complex ones like azo bonds. Finally, the 37°C temperature is consistent with the optimal range for Bacillus spp. to maximize enzyme activity, membrane fluidity, and metabolic processes (; ). During the biodegradation of contaminants, various physicochemical factors (pH, temperature, carbon source, dye concentration, and incubation period) directly influence the decolorizing capacity of microorganisms ().
FIGURE 5
Bacterial inoculation’s impact on plant development and dye decolorization in FTWs
To gauge the impact of the dye and bacterial inoculation on plant development, the root and shoot lengths of T. domingensis were measured at the conclusion of the experiment.
Dye-contaminated water has a significant effect (p < 0.05) on the plant growth, but bacterial inoculation enhanced root length (33%) and shoot length (13%) of the plant (Figure 6). T7 and T8, which combined the utilization of plants and bacteria, showed the greatest plant growth as compared to the other FTW-designed treatments. Despite the toxicity of dyes, these findings support the positive benefits of bacterial inoculation on plant development. Microorganisms play an essential role in plant growth and in the removal of toxic contaminants, thereby lessening the negative impact on the plant. This could be in response to the enzymatic activities that occur jointly in plants and bacteria to break down the dyes into simpler products. Additionally, under stress, numerous biochemical and molecular pathways are activated to enhance biological activities, such as phytohormone production and ACC deaminase activity ().
FIGURE 6
The percentage of dye decolorization of various treatments (T1–T9) after 30, 60, and 90 days is shown in Figure 7. The activity was found to be time-dependent as a consistent trend of increasing decolorization efficiency with time was observed for all the treatments. The treatments resulted in significant decolorization with the highest rate recorded in T7, nearly 90% at 90 days, and T8 also performed well with more than 80% decoloration. The findings are in line with those of previous studies that found bioaugmented FTWs more effective for dye decolorization than non-bioaugmented ones, attributed to the synergistic effect of plant roots and their associated microorganisms (; ).
FIGURE 7
Phytotoxicity assessment of treated water
A seed germination experiment was used to assess the harmful effects of azo dyes (SR, SY, and SB) treated in FTWs. Triticum aestivum seeds treated with water vs. dye-enriched water were compared in terms of seed germination, shoot, and root lengths (Table 1). According to the findings of the phytotoxicity bioassay, the FTW treatments created in this study considerably decreased the toxicity of the dye and its broken-down metabolites. The results align with those of earlier studies that demonstrated that treated wastewater from bioaugmented FTWs was less phytotoxic and resulted in significantly higher germination and growth of wheat seedlings (; ).
TABLE 1
| Treatments | % Germination | Shoot length (cm) | Root length (cm) |
|---|---|---|---|
| Tap water | 95 | 9.1 ± 0.81 | 5.1 ± 0.84 |
| C1 | 35 | 2.3 ± 0.04 | 1.14 ± 0.04 |
| C2 | 30 | 2.1 ± 0.20 | 1.43 ± 0.16 |
| C3 | 20 | 1.9 ± 0.09 | 0.9 ± 0.2 |
| T-1 | 85 | 7.16 ± 1.02 | 3.9 ± 0.64 |
| T-2 | 80 | 6.83 ± 1.43 | 2.5 ± 0.4 |
| T-3 | 50 | 5.33 ± 0.47 | 2.9 ± 0.41 |
| T-4 | 75 | 6.93 ± 0.09 | 3.26 ± 0.71 |
| T-5 | 80 | 6.66 ± 0.47 | 3.16 ± 1.31 |
| T-6 | 65 | 5.5 ± 1.47 | 2.17 ± 1.02 |
| T-7 | 90 | 8.9 ± 0.7 | 3.75 ± 0.40 |
| T-8 | 85 | 8.6 ± 0.61 | 3.46 ± 0.62 |
| T-9 | 70 | 6.06 ± 0.41 | 2.56 ± 0.41 |
Shows the effect of dyes, Bacillus sp., Typha domingensis and their synergistic interaction on the growth of Triticum aestivum.
Conclusion
The results showed that bioaugmented FTWs with T. domingensis and Bacillus sp. were effective and environmentally friendly approach for the treatment of textile effluent contaminated with azo-dye. Under optimal physicochemical conditions, the strain SZ1 of the genus Bacillus, out of six bacterial isolates, showed the highest ability in dye decolorization, especially in the removal of SR and SY. SZ1 was highly effective in dye removal, as high as 95% in FTWs, and better in plant growth parameters, showing decreased toxicity. This environmentally friendly, cost-effective technology is an effective substitute to traditional wastewater treatment methods for the treatment of industrial wastewater. This system is still to be validated at field scale and to assess its performance in long-term conditions in real textile effluents.
Statements
Data availability statement
The original contributions presented in this study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.
Author contributions
SZ: Resources, Methodology, Data curation, Writing – original draft. KF: Data curation, Methodology, Supervision, Writing – review & editing. MuA: Conceptualization, Writing – review & editing, Project administration. MJ: Software, Investigation, Writing – review & editing, Visualization. HI: Writing – review & editing, Methodology, Validation, Formal analysis. MaA: Validation, Data curation, Writing – review & editing, Resources. MoA: Resources, Funding acquisition, Visualization, Writing – review & editing. SI: Writing – review & editing, Supervision, Writing – original draft, Conceptualization. SM: Formal analysis, Project administration, Methodology, Writing – review & editing. IJ: Visualization, Funding acquisition, Resources, Writing – review & editing. HA: Resources, Funding acquisition, Software, Writing – review & editing. A-EF: Writing – review & editing, Resources, Software, Data curation, Visualization. AA: Visualization, Writing – review & editing, Investigation, Resources.
Funding
The author(s) declared that financial support was received for this work and/or its publication. The authors would like to acknowledge the Deanship of Graduate Studies and Scientific Research at Taif University for funding this work.
Conflict of interest
The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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Summary
Keywords
azo dye de-coloration, Bacillus sp., bioaugmentation, floating treatment wetlands, Typha domingensis
Citation
Zahid S, Fatima K, Ali M, javed M, Ibraheem H, Afzal M, Abohashrh M, Iqbal S, Mahmood S, Jafri I, Abdelmigid HM, Farouk A-E and Alyamani A (2026) Enhanced azo dyes decolorization in bioaugmented floating treatment wetlands using Typha domingensis and Bacillus sp.. Front. Microbiol. 17:1848974. doi: 10.3389/fmicb.2026.1848974
Received
10 April 2026
Revised
13 June 2026
Accepted
25 June 2026
Published
13 July 2026
Volume
17 - 2026
Edited by
Sikandar I. Mulla, REVA University, India
Reviewed by
Bhaskara Rao Kv, VIT University, India
Vishal Kumar Parida, Amity University Jharkhand, India
Updates
Copyright
© 2026 Zahid, Fatima, Ali, Javed, Ibraheem, Afzal, Abohashrh, Iqbal, Mahmood, Jafri, Abdelmigid, Farouk and Alyamani.
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Shahid Iqbal, Shahid.Iqbal@nottingham.edu.cn; shahidcbi8@outlook.com
Disclaimer
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