Advanced strategies for enhanced decolorization and detoxification of textile dyes using biofilm NAS₂–Ag/AgCl/Fe₃O₄ nanocomposites immobilized on peach pit

Abstract

As the most diverse prokaryotes, bacteria can decolorize dyes and produce nanoparticles. In this research, a mixture of four commonly used textile dyes, Reactive Red 120, Acid Red 18, Acid Red 137, and Direct Yellow 12, was effectively decolorized using three bacterial strains (S₂, N, and A) isolated from wastewater in textile facilities. These strains were applied as a consortium NAS₂, and in biofilm form, combined with an Ag/AgCl/Fe₃O₄ nanocomposite on a peach pit. Furthermore, strain S₂ was explicitly employed to synthesize the Ag/AgCl/Fe₃O₄ nanocomposite, which exhibits antimicrobial properties. The combination of four dyes was decolorized entirely by consortium NAS₂ within 24 h and by the biofilm NAS₂–Ag/AgCl/Fe₃O₄ nanocomposite (B-NAS₂-NC/PP) within 12 h, as confirmed by UV–Vis spectroscopy. FTIR analysis of the decolorized dye mixture revealed the breakdown of dye bonds. GC-MS analysis indicated that the bacterial strains degraded the dyes into more minor compounds, primarily decanoic acids and esters. SEM/EDX analysis of B-NAS₂-NC/PP showed successful stabilization of the bacterial strains and the nanocomposite on the peach stone. The Ag/AgCl/Fe₃O₄ nanocomposite was synthesized and characterized by UV–Vis, FTIR, XRD, and SEM, with an average nanocomposite size of 22 nm. The impact of the nanocomposite, dyes, and decolorized products was determined through radish seed germination and brine shrimp assays. Radish seed growth reached 77% in the presence of the nanocomposites, compared to 67% with the decolorized product. Notably, radish seeds failed to germinate in the presence of the four-dye mixture. The brine shrimp lethality rate was 11% at a nanocomposite concentration of 25 µg/ml, rising to 40% at 2500 µg/ml. The decolorized product at a 1250 µg/ml concentration of the dye mixture showed a 0% lethality rate.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-026-40451-4.

Introduction

The textile industry, with its global reach and advanced technological foundation, plays a vital role in the world economy. However, its intensive production and extensive resource consumption have led to considerable environmental degradation, posing serious risks to ecosystems and various forms of life¹.

The most noticeable environmental damage caused by the textile industry results from the discharge of highly colored, untreated effluents into receiving bodies, which are carcinogenic².

Textile wastewaters render water and soil unsuitable for use in areas near disposal sites. This may reduce light penetration to the lower parts of the water, adversely affecting photosynthesis and aquatic flora and posing a long-term threat to biodiversity^3,4.

Synthetic dyes have chemically diverse origins and molecular structures. These organic compounds, mainly categorized into different groups such as direct, reactive, and acidic dyes, are long-lasting colorants and are significantly soluble in water. They exhibit high stability in water, posing a challenge for treatment using traditional systems^5,6.

Various biological, chemical, and physical processes, such as activated carbon, photooxidation, adsorption, and coagulation, are used to remove effluent dyes⁷. Some of these systems have proven to be effective. However, the biosystem may need to improve in application, particularly in high-energy-demand applications, in the formation of toxic metabolites, and in secondary disposal problems due to the generation of a significant amount of sludge. This results in a new pollution issue requiring further treatment⁸. Among all the mentioned techniques, bio-processing approaches are developing rapidly and can overcome these defects due to their cost-effectiveness and environmentally benign nature^8,9.

Biotransformation of dye-containing wastewater into safe by-products is a promising way to clean up dye-contaminated environments⁷. Various microorganisms within microbial populations, including bacteria, fungi, and yeast, have been studied for their capacity to decolorize a broad spectrum of synthetic dyes^10,11. Bacterial decolorization, in particular, has increasingly captured scientists’ interest, primarily due to its lower cost, simplicity, and high efficiency associated with various oxidoreductive enzymes, which are practical both intracellularly and extracellularly^8,12.

The use of bacteria for dye removal is often limited by the excessive biomass generated during treatment. Immobilization of bacterial cells has emerged as a highly effective strategy, stabilizing cells, enabling recovery and reuse, and thereby reducing overall operational costs. Moreover, this approach facilitates efficient dye removal from effluents without producing additional toxic by-products¹³.

Recent advances in microbial biofilms and immobilized cell systems further enhance the potential of bacterial treatments compared to free-cell systems^14,15. Biofilms, which naturally develop on solid surfaces¹⁶, offer mechanical stability, operational resilience, and the ability to reuse biomass¹⁷. Importantly, biofilms promote interactions between microbial cells, enabling metabolic activities that are often more diverse, efficient, and economically advantageous than those of free-living cells¹⁸.

Biofilms facilitate unique cellular interactions, giving rise to diverse behaviors and economically important activities that are distinct from those of free-living cells¹⁸. Nanotechnology has emerged as a highly effective approach for water treatment, with nanoparticles serving as efficient nano-adsorbents¹⁹. The synthesis of nanoparticles encompasses a wide array of physical, chemical, and biological methods, ranging from conventional techniques to innovative strategies^19,20. Recent advances in green chemistry offer clean, non-toxic, and eco-friendly approaches that enhance nanoparticle production, including the use of bacteria, fungi, and plants for biogenic synthesis²⁰.

Recent advances in nanotechnology have enabled the use of various nanoparticles for the degradation of azo dyes and wastewater purification²¹. Among these, silver nanoparticles (AgNPs) have attracted significant attention due to their versatile applications in catalysis, sensing, antibacterial activity, and water treatment²². To develop effective strategies for mitigating the harmful effects of these dyes, it is crucial to evaluate the toxicity of both the dyes and their degradation products¹³.

The advancement of nanotechnology has enabled the use of nanoparticles for the degradation of azo dyes and the purification of wastewater²¹. Among these, silver nanoparticles (AgNPs) have gained significant attention due to their diverse applications in catalysis, sensing, antibacterial treatments, and water purification²¹. Evaluating the toxic properties of dyes and their degradation by-products is essential for developing strategies to minimize their environmental impact¹³. In this context, the acute toxicity test using Artemia salina serves as a crucial tool for assessing the potential toxicity of by-products generated during the biodegradation process^23,24.

This research uses a nanocomposite of Ag/AgCl/Fe₃O₄ using a thermotolerant bacterial strain S₂. The decolorization of a mixture of four dyes commonly used in the textile industry is then investigated with two biological systems: a microbial consortium, NAS₂, and a biofilm, NAS₂, combined with the nanocomposite, stabilized within the pores of peach pits. Furthermore, the toxicity of the decolorized products is assessed on living organisms, including bacteria, brine shrimp, and radish seeds.

Materials and methods

Selection of dye-degrading bacteria

This study used fourdyes from textile factories: Acid Red 137, Acid Red 18, Direct Yellow 12, and Reactive Red 120. Three bacterial strains, designated N, A, and S₂, were isolated in the microbiology laboratory of Kashan University for dye decolorization. Strain A, isolated from textile wastewater, is a Gram-positive coccus with 100% similarity to Enterococcus casseliflavus. Strain N, also isolated from textile wastewater, is a Gram-positive bacillus belonging to the genus Marinospirillum, with a 97.24% similarity to Marinospirillum alkaliphilum²⁵. Strain S₂, isolated from Choghart iron ore soil, shows a 99.68% similarity to Bacillus zhangzhouensis. This bacterium is a Gram-positive coccus and hyperthermophilic (heat-resistant)²⁶.

Dye-removing bacterial consortium NAS₂

The bacterial consortium NAS₂, comprising strains N, A, and S₂, was studied for decolorization of a mixture of four dyes in nutrient broth. Strains N, A, and S₂ were individually cultured on nutrient agar and inoculated into nutrient broth after 24 h. When the turbidity of the bacterial suspensions of strains N, A, and S₂ reached half the McFarland standard, equal volumes of the three suspensions were mixed to form the bacterial consortium. Acid Red 137, Direct Yellow 12, Acid Red 18, and Reactive Red 120 at 50 ppm were inoculated into test tubes containing 10 ml of nutrient broth and 1% inoculum from a half McFarland standard. The above media were then incubated in a shaker incubator at 130 rpm and 30 ⁰C. At intervals of 12, 24, 36, and 72 h of incubation, 1.5 ml of the suspension was separated by centrifugation at 7000 rpm for 10 min. The supernatant was then analyzed using ultraviolet light and a spectrophotometer. The percentage of decolorization was calculated using the following formula²⁵

where:

A₀ is the absorbance of the medium after decolorization,

A is the absorbance of the control medium,

D is the percentage of decolorization.

Decolorization of biofilm NAS₂–Ag/AgCl/Fe₃O₄ immobilization on peach stone

The sterilized peach pits were added to nutrient broth containing the bacterial consortium NAS₂ and incubated at 30 °C for seven days. Following incubation, the pits were transferred to a decolorizing medium and evaluated by spectrophotometry for 12 h.

To extract the decolorization products from the four-color mixture, the decolorizing medium was centrifuged at 6000 rpm for 10 min, and the supernatant was separated. The supernatant was then transferred to a decanter containing an equal volume of ethyl acetate solution, and the mixture was shaken to mix the two phases. The aqueous phase was discarded after the phases were separated for 30 min. This process was repeated three times. Finally, the remaining solution was dried, and the resulting powder was analyzed using GC-MS and FTIR.

Ag/AgCl/Fe₃O₄ composite-producing bacteria

In this study, strain S₂ was employed to synthesize the Ag/AgCl/Fe₃O₄ nanocomposite. The supernatant from the cultivation of strain S₂ was centrifuged at 7000 rpm for 20 min at 4 °C after 24 h. Fe₃O₄nanoparticles were synthesized using strain S₂ following the method described by Eshghi et al. in 2022. A mixture of 3 mM silver nitrate and 0.0399 g of Fe₃O₄ nanoparticles was prepared in 100 ml of sterilized deionized water, to which 40 ml of bacterial supernatant was added. The resulting mixture was then exposed to light, and its properties were evaluated at 2, 4, and 6 h using a spectrophotometer. The synthesized composite was subsequently centrifuged, separated, and dried. The characteristics of the nanocomposites were analyzed using FTIR, XRD, and SEM/EDX techniques. Additionally, the stability of the Ag/AgCl/Fe₃O₄ nanocomposite with bacteria on the surface of peach kernels was assessed for its decolorization capability.

Antimicrobial properties of Ag/AgCl/Fe₃O₄ composite

The antimicrobial activity of the synthesized nanoparticles was evaluated against standard microbial strains Pseudomonas aeruginosa (ATCC 27,853), Staphylococcus aureus (ATCC 29,737), Staphylococcus epidermidis (ATCC 11,228), Candida albicans (ATCC 10,231), and strains N, A, and S₂ using the well diffusion agar technique by CLSI 2024 guidelines²⁶.

Toxicity against living organisms

Brine shrimp

The toxicity of the dye samples and the Ag/AgCl/Fe₃O₄ nanocomposite was evaluated using the brine shrimp lethality test. Brine shrimp eggs were obtained from an American company named City, 84,126 Utah Inc., Salt Lake. The experimental procedure was as follows:

One liter of saltwater was prepared to hatch the brine shrimp eggs into larvae. For the saltwater preparation, 23 g of sodium chloride, 11 g of magnesium chloride, 4 g of sodium sulfate, 1.3 g of calcium chloride, and 0.7 g of potassium chloride were dissolved in water. The pH of the solution was adjusted to 9 with sodium carbonate. Then, 0.1 g of brine shrimp eggs were transferred into the solution inside an aquarium container and placed in a water bath at 30 °C for 48 h, with aeration provided by an air pump and illumination from a lamp.

Test tubes containing 10 larvae each were exposed to concentrations of 25, 250, 750, 1250, 1750, and 2500 µg/ml of each of the four dyes: Acid Red 137, Acid Red 18, Direct Yellow 12, and Reactive Red 120, and Ag/AgCl/Fe₃O₄nanocomposite, as well as the product obtained from the decolorization process. The test tubes were maintained at 30°C under appropriate light conditions for 24 h. The numbers of live and dead larvae and the lethality percentage were calculated using (Eqs. (1) and (2))²⁷. The experiment was repeated three times to ensure accuracy.

1

m: Average number of dead larvae in the sample.

M: Average number of dead larvae in the control.

S: Average number of live larvae in the control.

Equation (2) Killing Percentage Equation

2

Based on the above formulas, a graph showing the percentage of mortality as a function of concentration (µg/ml). From this graph, the LC₅₀ value, which is the concentration of the sample that can kill 50% of the shrimp, was calculated in µg/ml.

Radish seeds

The toxicity levels of four dyes, the decolorization product, and the nanocomposite were evaluated using radish (Raphanus sativus L.) seeds. All materials were exposed to UV radiation under a laminar hood before testing. Radish seeds were purchased from a local market in Kashan, Iran. The seeds were surface-sterilized by sequential washing: 10 min in sterile distilled water, 1 min in 70% ethanol, and 5 s in sodium hypochlorite solution, repeated three times. The sterilized seeds were placed in plates and treated with the dye mixture, the decolorization product, the nanocomposite, and distilled water (control). After 7 days of incubation under natural sunlight, seed growth (leaves, stems, and roots) was measured, and the results were evaluated using the specified equations (3), (4), and (5)²⁶.

Equation (3) for determining Relative Seed Germination (RSG)

3

Equation (4) relative root growth RRG

4

Equation (5) germination index GI

5

Bacteria

E. coli ATCC 10,536 was culturedon nutrient agar supplemented with the dye mixtures at concentrations of 11, 22, 33, and 44 µg/ml and the degradation product. After 24 h of incubation, the absorbance was measured with a spectrophotometer and compared with a control sample lacking the dyes.

Results and discussion

Nanocomposite synthesis and characterization

This study investigates the green synthesis and characterization of an Ag/AgCl/Fe₃O₄ nanocomposite using the cell free supernatant from B. zhangzhouensis strain S₂. The approach led to a distinct color change and the successful formation of the composite. UV–Vis spectroscopy and X-ray diffraction (XRD) analyses confirmed the presence of nano-silver, AgCl, and magnetic iron oxide components, underscoring the effectiveness of this bacterial-mediated bioreduction process.

The cell-free supernatant from strain S₂ was added to a mixture of AgNO₃ and Fe₃O₄ nanoparticles, resulting in an immediate color change. As illustrated in (Fig. 1A), the solution turned completely black after 6 h, signaling nanoparticle formation. Subsequent UV–Vis analysis confirmed the synthesis of the Ag/AgCl/Fe₃O₄ composite, with characteristic absorption peaks indicative of silver surface plasmon resonance and contributions from the iron oxide phases.

Fig. 1.

Evaluation of Ag/AgCl/Fe₃O₄ nanocomposites results using UV–Visible (A) after 2 h (a), 4 h (b), and 6 h (c) formed by strain S₂, as well as AgNO₃ solution (d) and FeSO₄ solution (e). The analysis also includes XRD (B), FTIR (C), EDX (D), and SEM (E).

The crystalline structure and phase composition of the biosynthesized nanoparticles were examined using XRD (Fig. 1B). Diffraction peaks were compared against standard reference patterns from the Joint Committee on Powder Diffraction Standards (JCPDS). Prominent peaks at approximately 27.8°, 32.2°, 46.3°, and 54.8° (2θ) corresponded to the characteristic reflections of silver chloride (AgCl, JCPDS card No. 00-006-0480), confirming the bacterial-mediated formation of highly crystalline AgCl nanoparticles. Peaks at around 35.4°, 57.3°, and 62.9° aligned with magnetite (Fe₃O₄, JCPDS card No. 01-075-0033), suggesting transformation of iron ions by the bacterial medium. Additionally, low-intensity peaks matching JCPDS card No. 01-087-0717 indicated a minor metallic silver (Ag⁰) phase, likely resulting from partial bioreduction of Ag⁺ ions through bacterial metabolites, such as enzymes or extracellular polymeric substances. Notably, no peaks attributable to sodium chloride (NaCl, JCPDS No. 01-088-2300) were detected, implying its absence or levels below the detection threshold. The average crystallite size of these cubic nanoparticles, calculated using the Debye–Scherrer equation, was approximately 16.42 nm, indicating their nanoscale dimensions and high crystallinity.

Fourier Transform Infrared (FTIR) spectroscopy provided further insights into the functional groups involved (Fig. 1C). The absorption peak at 1636.11 cm⁻¹ was attributed to H–O–H bending vibrations or C=O stretching in amide I groups, commonly associated with proteins, thereby supporting their role in nanoparticle reduction and stabilization. A distinct band at 1124.45 cm⁻¹ corresponded to C–O stretching vibrations, indicative of alcohols, ethers, or polysaccharides derived from bacterial extracellular polymeric substances or cell wall components. Furthermore, the peak at 553.95 cm⁻¹ was attributed to metal–oxygen (M–O) stretching vibrations, confirming the presence of metal or metal oxide nanoparticles, potentially involving Fe–O or Ag–O bonds. Collectively, these FTIR findings confirm the success of the eco-friendly synthesis and underscore the pivotal role of bacterial biomolecules in metal-ion reduction and nanoparticle stabilization.

Energy-dispersive X-ray spectroscopy (EDX) analysis (Fig. 1D) further corroborated the successful biosynthesis of silver nanoparticles (AgNPs). The elemental composition revealed silver (Ag) as the predominant element at approximately 76.10 wt%, reflecting the efficiency of the bacterial synthesis. Carbon (17.76 wt%) and oxygen (3.62 wt%) were also detected, likely originating from organic residues such as bacterial secretions or biomolecules serving as surface capping agents. A trace amount of iron (2.53 wt%) was identified, possibly from culture medium contaminants, instrumentation, or sample preparation. Detection confidence levels (Class A for Ag, C, and O; Class B for Fe) confirmed the reliability of these assignments. ZAF (atomic number, absorption, and fluorescence) correction factors were applied to enhance quantitative accuracy, yielding a ZAF value of 0.8953 for silver.

Overall, the EDX results in (Fig. 1D) offer compelling evidence for the biosynthesis of silver nanoparticles characterized by high purity and minimal contamination. The presence of carbonaceous and oxygenated species suggests surface functionalization with biological moieties, potentially enhancing stability, dispersibility, and bioactivity in subsequent applications. This functionalization could involve adsorption of bacterial proteins or polysaccharides, potentially reducing agglomeration and improving biocompatibility.

Surface morphology and microstructural features of the biosynthesized nanoparticles were assessed using field emission scanning electron microscopy (FE-SEM), as shown in (Fig. 1E). The images revealed nanoscale particles with a predominantly spherical to slightly irregular morphology. Some aggregation was observed, likely attributable to high surface energy and the stabilizing influence of biogenic capping agents. Despite this, the particles displayed reasonable uniformity in size and distribution. The average particle size remained within the nanometer range (< 100 nm), consistent with XRD-derived crystallite sizes. High-resolution imaging indicated closely packed crystallites, reinforcing the crystalline nature observed in XRD.

Such packing and moderate agglomeration may arise from interactions among biomolecules secreted by the bacteria, which function as both reducing and stabilizing agents. In essence, FE-SEM analysis substantiates the bacterial-mediated synthesis of nanoparticles exhibiting favorable nanometric dimensions, dispersion, and morphologies suitable for applications in antimicrobial, catalytic, or biomedical contexts. Based on SEM results from (Fig. 1E), the cubic Ag/AgCl/Fe₃O₄ nanocomposite sizes ranged from 7 to 21 nm. The size distribution was analyzed and plotted using Digimizer software (Figure S1).

As reported by Padilla-Cruz et al. (2021), FTIR analysis of bimetallic Fe/Ag nanoparticles displayed characteristic peaks at 3400 cm⁻¹, 2920 cm⁻¹, 1640 cm⁻¹, 1430 cm⁻¹, and 1080 cm⁻¹. The band in the 3400–3000 cm⁻¹ range corresponds to O–H stretching vibrations from hydroxyl groups, while the 2920 cm⁻¹ peak relates to aliphatic C–H stretching. The peaks at 1640 cm⁻¹ and 1430 cm⁻¹ are attributable to C=O and C=C stretching, respectively, and the 1080 cm⁻¹ peak to C–O stretching²⁸. These observations align with our FTIR data, implying comparable biomolecular involvement in nanoparticle capping and stabilization, possibly through hydrogen bonding or electrostatic interactions.

Disk diffusion assays demonstrated notable antimicrobial activity of the nanocomposite against standard bacterial strains, with inhibition zone diameters ranging from 9 to 18 mm at concentrations of 0.02–10 mg/ml (Figure S2). Bactericidal effects were apparent at 0.5–10 mg/ml across all strains. In contrast, no such activity was observed at 0.02 mg/ml for strains N, A, and S₂, rendering this concentration suitable for dye decolorization in combination with the bacterial consortium. These findings underscore the dual functionality of the nanocomposite, which acts not only as an effective antimicrobial agent but also as a facilitator of dye removal. This bifunctional performance is likely mediated through mechanisms such as the generation of reactive oxygen species or the disruption of cellular membranes. Consequently, the nanocomposite not only supports dye decolorization but also shows potential for wastewater disinfection in dyeing processes, integrating contaminant removal with microbial control to improve treatment efficiency. Nevertheless, limitations including potential toxicity at elevated concentrations, scalability for industrial applications, and long-term environmental impacts (e.g., silver ion release) merit further exploration.

In a related eco-friendly synthesis, bimetallic silver and iron oxide nanoparticles derived from Adansonia digitata L. fruit shell extract exhibited strong antifungal activity against tomato fungi²⁹. This work parallels ours in demonstrating antimicrobial synergy within bimetallic systems, although our emphasis lies on bacterial targets and dye degradation. Another study involving Ag–Fe bimetallic nanoparticles derived from Gardenia jasminoides leaves reported potent antimicrobial properties and enhanced synergy against drug-resistant strains compared with monometallic variants²⁸. Such synergy resonates with our observed bactericidal effects, likely mediated by improved electron transfer at the Fe/Ag interface.

Recent investigations have leveraged bacterial strains to produce nanocomposites for various applications. In 2021, Staphylococcus pasteuri sp. nov. ZAR1 was employed to generate an Ag/AgCl nanocomposite with antimicrobial attributes³⁰. By 2022, Bacillus paralicheniformis Tmas-1 yielded a comparable nanocomposite effective in decolorizing Rhodamine B. A further study utilized B. zhangzhouensis S₂ to produce magnetic iron nanoparticles that degraded Acid Red 88 dye and mitigated its toxicity when encapsulated in an alginate matrix³¹. These bacterial-driven syntheses align with our methodology, yet our nanocomposite distinctively incorporates disinfection capabilities, positioning it as a multifunctional tool for wastewater remediation.

In one investigation, an Ag/AgCl/Fe₃O₄ nanocomposite was chemically synthesized as a recoverable magnetic disinfectant for wastewater. It effectively eradicated E. coli K-12 and exhibited high efficacy in treatment processes³². In contrast to our biological approach, this chemical method prioritizes recyclability through magnetic separation, suggesting avenues for hybrid strategies to optimize sustainable production and practical recovery.

Dye degradation

In this study, we employed four azo dyes commonly encountered in textile wastewater: Reactive Red 120, Direct Yellow 12, Acid Red 137, and Acid Red 18. These dyes were selected due to their widespread use and environmental persistence, underscoring the need for effective removal strategies. Spectrophotometric analysis revealed distinct absorption maxima for each: Reactive Red 120 at 539 nm, Direct Yellow 12 at 318 nm, Acid Red 18 at 507 nm, Acid Red 137 at 501 nm.

Consortium NAS₂

Initially, a mixture of the four dyes at 50 mg/L was tested against the individual strains N, S₂, and A. After 24 h, all strains effectively eliminated the dye absorption peaks. As shown in (Fig. 2A), strain A achieved the highest decolorization rate (99%), while strain N showed the lowest (94%). Moreover, the NAS₂ consortium was evaluated, achieving 97% decolorization within 24 h (Fig. 2B).

Fig. 2.

Evaluation of the percentage of growth and decolorization of four-dye mixture by strains A, N (A), and S₂ and consortium NAS₂ (B) after 24 h.

Microbial consortia have emerged as robust tools for degrading textile dyes in wastewater, often outperforming single strains through synergistic interactions. For instance, a consortium of Bacillus subtilis, Brevibacillus borstelensis, and Bacillus firmus degraded Reactive Red 170, producing extracellular polymeric substances (EPS) and biofilms that rendered the metabolites non-toxic³³. In another approach, bioreactors employing sludge, alkalophilic, and thermophilic consortia achieved 85–94% azo dye removal, with the thermophilic variant reaching 93.5% decolorization and the alkalophilic one reducing chemical oxygen demand (COD) by 93%³⁴. Similarly, Das and Mishra (2017) reported that a consortium of Zobellella taiwanensis and Bacillus pumilus decolorized Reactive Green-19 by over 97% within 24 h, following first-order kinetics³⁵. These examples illustrate how consortia enhance efficiency via complementary enzymatic activities, such as azoreductases and laccases, which cleave azo bonds and facilitate mineralization. Consortium NAS₂ aligns with this study, but innovatively integrates an Ag/AgCl/Fe₃O₄ nanocomposite synthesized by strain S₂, accelerating degradation through photocatalytic synergy while enabling antimicrobial control.

Biofilm NAS₂–Ag/AgCl/Fe₃O₄ immobilized on peach pit

The biofilm formed by the three-strain consortium, combined with the nanocomposite on a peach pit substrate, degraded the dye mixture by 91% within 12 h, even though the growth of the bacteria responsible for the biofilm was 40% in the presence of the dye mixture (Fig. 3A).

Fig. 3.

Decolorization results of the four-dye mixture by Biofilm NAS₂–Ag/AgCl/Fe₃O₄ immobilized on peach pit after 12 h: growth and decolorization percentage, UV–Vis results (A), EDX analysis of B-NAS₂-NC/PP (B), and SEM image of the biofilm of strains N, A, and S₂ with nanocomposites trapped in peach-pit pores (C).

The biofilm formed by the three-strain consortium, along with the nanocomposite on a peach pit substrate, reduced the dye mixture by 91% within 12 h, even though the growth of the bacteria responsible for the biofilm was only 40% in the presence of the dye mixture. These results indicate that biofilm bacteria and nanoparticles on peach pits present a promising method for environmental pollutant removal. The study also demonstrated biofilm-associated cells are more efficient at degrading dyes than free cells, highlighting biofilms as an environmentally friendly solution.

Various studies have investigated the removal of dyes using bacterial biofilms. Haque et al. (2022) identified ten novel biofilm-producing bacteria capable of decolorizing Direct Red 28 with 97.8% to 99.7% efficiency under optimal conditions. The reduction in COD and increased seed germination indicated that the biodegraded products were less toxic³⁶.

Free-floating bacterial cells can decolorize Congo Red but often produce toxic compounds and are more vulnerable to pollutants, while biofilms are more resistant. In another study, four novel bacterial biofilm consortia were developed, effectively decolorizing CR within 72 h. UV–Vis and FTIR analyses showed that the dye’s main peaks had disappeared or changed, and the decolorized products were non-toxic, making these consortia suitable for bioremediation³⁷.

Silver nanoparticles synthesized by strain N were immobilized in a calcium alginate matrix and utilized to decolorize Disperse Blue 183 dye²⁵. This study examined the biodegradation of pesticides (cypermethrin and imidacloprid) and dyes (Malachite green and Congo red) using bacterial biofilms from contaminated soils and dye effluents. Different bacterial biofilms showed varying effectiveness, with some achieving high degradation rates for both pesticides and dyes³⁸.

SEM/EDX of NAS₂–Ag/AgCl/Fe₃O₄ nanocomposite consortium stabilized on peach stone

Immobilization of the nanocomposite and biofilm NAS₂ on peach stone enabled effective dye removal. SEM images (Fig. 3C) showed bacterial cells adhering to the porous surface, both in clusters and individually, as they naturally bonded during incubation. EDX analysis (Fig. 3B) confirmed the presence of significant iron and silver signals, verifying successful nanocomposite anchoring.

Comparable studies include Mehrzad et al. (2023), in which the dye-degrading strain N immobilized on titanium oxide nanoparticles decolorized Basic Blue 41, and SEM confirmed nanoparticle attachment to bacterial surfaces²¹. Another involved Enterobacter cloacae immobilized in calcium alginate for the decolorization of Reactive Blue 19, with GC-MS and FTIR confirming degradation and elimination of toxicity³⁸. Effective decolorization of a mixed solution of Reactive Blue 221, Reactive Yellow 145, and Reactive Red 195 was achieved within 192 h by bacterial consortia immobilized on polyethylene mesh, as confirmed by SEM analysis³⁴. These findings resonate with ours, where porosity of peach stone enhances mass transfer and protects against dye toxicity, though long-term stability in real effluents remains to be tested.

FTIR

Comparing FTIR spectra of the dye mixture and decolorized product (Fig. 4) revealed notable shifts. The untreated mixture (Fig. 4a) showed peaks at 3431.46 cm⁻¹ (O–H stretching), 2929.11 cm⁻¹ (C–H in alkanes), 1645.20 cm⁻¹ (C=C or C=O), and others linked to C–O and aromatic groups. In the decolorized product (Fig. 4b), similar peaks persisted (e.g., 3443.94 cm⁻¹ for O–H, 2924.98 cm⁻¹ for C–H, 1668.21 cm⁻¹ for C=C/C=O). However, the 883.42 cm⁻¹ aromatic peak vanished, indicating breakdown of aromatic and carbonyl structures while preserving core functional groups.

Fig. 4.

FTIR spectra of the mixture of four dyes before (a) and after (b) decolorization using B-NAS₂-NC/PP treatment.

Guo et al. (2021) observed similar changes in Metanil Yellow G decolorization by a thermophilic consortium, with disappearance of the 1591 cm⁻¹ azo bond peak, increased N–H signals (3300–3500 cm⁻¹), and sulfonic acid removal. Analogous FTIR shifts have been reported in halo-thermophilic consortia³⁹. These alterations suggest enzymatic cleavage of azo bonds, leading to less toxic amines or carboxylic acids, consistent with our reduced phytotoxicity and cytotoxicity.

GC-MS

During GC-MS analysis of the decolorization products generated by consortium NAS₂ in the presence of Ag/AgCl/Fe₃O₄ nanocomposite stabilized on peach stone, multiple distinct peaks were detected, each corresponding to specific degradation fragments of the dye mixture. This experiment involved four azo dyes with varying molecular weights: Reactive Red 120 (1338.1 g/mol), Acid Red 18 (604.48 g/mol), Acid Red 137 (464.43 g/mol), and Direct Yellow 12 (659.7 g/mol). The results showed that these products had substantially lower molecular weights than the original compounds (Fig. 5), indicating effective fragmentation into smaller molecules, likely through enzymatic cleavage of azo bonds and aromatic rings.

Fig. 5.

GC-MS Analysis of the product resulting from the decolorization of a mixture of four dyes by B-NAS₂-NC/PP.

Guo et al. (2021) reported similar findings in the decolorization of Metanil Yellow G by the thermophilic consortium HT1, identifying five intermediates via GC-MS that indicated azo bond cleavage and subsequent mineralization via the tricarboxylic acid (TCA) cycle³⁹. Likewise, Das and Mishra (2017) degraded Reactive Green 19 using a bacterial consortium, with chromatographic analysis revealing simpler metabolites, including long-chain aliphatic hydrocarbons, unsaturated carboxylic acids, and organic sulfides³⁵. These studies highlight the biodegradation of complex dyes into benign compounds, mirroring our observations where the nanocomposite likely enhances reductive cleavage via reactive oxygen species or electron transfer, accelerating the process.

The discharge of textile dyes into industrial wastewater poses irreversible environmental risks, harming aquatic life and exacerbating water scarcity. Given the diversity of dye structures, their removal is essential to safeguard ecosystems and human health. Biological methods, leveraging microorganisms for cost-effective, eco-friendly treatment, excel through processes such as biosorption, bioaccumulation, and metabolite formation that reduce or eliminate toxicity. Integrating these with nanoparticle technology, as in our approach, markedly boosts efficiency by combining microbial enzymes with photocatalytic activity. Our results demonstrate the power of bacterial consortia combined with the nanocomposites to decolorize and detoxify effluents, offering a sustainable path for wastewater management. Optimizing nanocomposite formulations, such as tuning particle size or loading, and testing against broader pollutants in real-world settings, addresses scalability challenges and environmental concerns, such as potential silver-ion leaching, paving the way for practical industrial adoption.

Biotoxicity

Based on the results of the brine shrimp toxicity assay, the toxicity profiles of the untreated four-dye mixture, Ag/AgCl/Fe₃O₄ nanocomposite (Fig. 6 A), the decolorization products generated by the bacterial biofilm consortium, and the nanocomposite immobilized on peach-stone biomass were systematically assessed. As illustrated in (Fig. 6B)the untreated four-dye mixture induced shrimp mortality rates generally exceeding 46% at concentrations of 250 µg/ml and above, with mortality increasing dose-dependently up to approximately 70–80% at 2500 µg/ml. The LC₅₀ value for the untreated mixture was determined to be 250 µg/ml. In contrast, the decolorization products from the bacterial biofilm and nanocomposite treatments showed markedly lower toxicity, with mortality ranging from 7% at 25 µg/ml to 23% at 2500 µg/ml, demonstrating a substantial reduction in toxicity post-decolorization.

Fig. 6.

Brine shrimp test for Ag/AgCl/Fe₃O₄ nanocomposites (A) and the four-dye mixture and the decolorization product of the four-dye mixture by B-NAS₂-NC/PP (B).

Although Ag/AgCl/Fe₃O₄ nanocomposite elicited up to 40% mortality at higher concentrations, the integrated operational system effectively mitigates environmental risks. This is achieved through immobilization of the nanocomposite on peach-stone biomass, retention within biofilm matrix NAS₂, and complete magnetic recoverability. These features collectively prevent nanoparticle dispersion into the environment and minimize Ag⁺ ion release. Notably, the treated effluent exhibited no acute toxicity (mortality < 5%), underscoring the system’s environmental safety under practical conditions and its favorable risk–benefit profile.

In a similar study, silver nanoparticles produced using leaf, stem, and fruit peel extracts of Prunus mahaleb showed low toxicity in the brine shrimp assay⁴⁰.

Comparative analysis of toxicity data for untreated dyes decolorized products, as depicted in (Fig. 6B)confirms that the nanobiological treatment significantly attenuates the toxicity of the dye mixture. These results highlight the efficacy of nanobiological systems in mitigating the adverse effects of textile dyes, thereby addressing key environmental concerns posed by industrial dye pollutants.

This aligns with prior research indicating that, while untreated textile dyes such as Congo Red and Direct Red 22 are highly toxic, their decolorized metabolites are considerably less harmful. For instance, Babu et al. (2015)⁴¹, Prasad and Rao (2013)⁴², and Nazari et al. 2024⁴³ documented reduced toxicity in decolorized dye products. More recent investigations have also assessed nanoparticle toxicity; Nazari (2021)²⁵ reported over 50% brine shrimp mortality from silver nanoparticles at 1 µg/ml, and Eshghi (2022)²⁶ observed comparable lethality with iron nanoparticles. However, studies on treatments combining iron nanoparticles with Ca-Alg beads for Acid Red 88 showed a dramatic reduction in toxicity to 10% for the decolorized form, compared to over 65% for the parent dye.

Complementary toxicity evaluations, including phytotoxicity and cytotoxicity assays, further verified that the degraded products were far less harmful than the original dyes, reinforcing the eco-friendly attributes of this approach. Nonetheless, ongoing challenges such as fine-tuning enzyme expression across varying pH and temperature regimes necessitate additional research to facilitate industrial-scale implementation.

Radish seed germination

The growth of radish seeds was examined in the presence of dye mixture, the decolorization product, and Ag/AgCl/Fe₃O₄ nanocomposite. (Fig. 7A) indicates that only 3% of the seeds germinated upon exposure to the dye mixture, highlighting its severe toxicity. Conversely, treatment with the decolorization products obtained from B-NAS₂-NC/PP significantly enhanced seed germination to 67%, while exposure to the Ag/AgCl/Fe₃O₄ nanocomposite powder resulted in an even higher germination rate of 77%. Furthermore, (Fig. 7B) clearly demonstrates the seed toxicity compared to the Germination Index (GI).

Fig. 7.

Phytotoxicity evaluation on radish seeds after one week of sunlight exposure: A Image of radish seed growth in the presence of the four-dye mixture, decolorization products, Ag/AgCl/Fe₃O₄ nanocomposites, and water as a control, after one week of exposure to light B Comparison of RSG and RRG growth metrics control (water), Mixture of four dyes, Decolorization products, Ag/AgCl/Fe₃O₄ Nanocomposites.

Several studies have demonstrated that microbial treatments can effectively decolorize dyes and reduce their toxicity. Seyedi et al. showed⁴⁴ that bacterial strains isolated from textile wastewater completely eliminated the toxicity of Disperse Blue 60 dye in radish seeds after decolorization. Another study found that the fungal strain Coriolopsis gallica BS9, which produces laccase enzymes, efficiently decolorized the poly-azo dye Sirius Grey in the presence of 1-hydroxybenzotriazole (HBT), reducing its phytotoxicity. Additionally, a cell-free supernatant containing laccase from the same fungal strain, combined with HBT, achieved 87.56% decolorization of Sirius Grey and decreased its toxicity⁴⁵.

In contrast, the results indicate that Acid Red 88 exhibits substantial toxicity, markedly inhibiting seed growth, whereas normal growth was observed following its degradation by Cu nanoparticles stabilized on Marinospirillum alkaliphilum strain N⁴⁶.

Bacteria growth

The toxic effects of varying concentrations of dye mixture on E. coli were evaluated using spectrophotometric measurements of bacterial growth. Exposure to the dye mixture resulted in concentration-dependent reductions in growth rates. Specifically, after 24 h of incubation, optical density readings indicated significantly lower bacterial proliferation at concentrations of 11, 22, 33, and 44 mg/l compared to the untreated control group. This pattern suggests that the dye mixture inhibits E. coli growth. In contrast, cultures exposed to the decolorization products exhibited growth rates comparable to those of the control, indicating successful detoxification of the dyes via degradation.

Supporting these observations, prior research on dye decolorization underscores the efficacy and safety of bioremediation approaches. For instance, one investigation showed that the decolorization of Disperse Blue 183 using silver nanoparticles combined with bacterial cells immobilized in alginate beads yielded degradation products that were non-toxic in bacterial assays, in contrast to the antimicrobial properties of the dye⁴⁷. Similarly, degradation of malachite green by Kocuria marina produced metabolites that lacked the inhibitory effects of the pure dye on bacterial growth⁴⁷. Furthermore, Lysinibacillus sphaericus D3 immobilized in sodium alginate beads achieved effective decolorization of xylidine orange, with the resulting products demonstrating no toxicity in disc diffusion tests against 12 marine bacterial strains, unlike the untreated dye⁴⁸. These findings indicate that such microbial and nanomaterial-based methods can safely degrade dyes, potentially mitigating environmental risks associated with textile effluents.

Conclusion

Overall, this research highlights the potential of using bacterial consortia and nanocomposite materials to effectively decolorize and detoxify industrial textile dye effluents, thereby promoting sustainable wastewater treatment practices. The integration of microbial and nanotechnological approaches opens new avenues for the efficient and eco-friendly management of industrial pollutants. The bacterial consortium NAS₂ demonstrated remarkable decolorization capabilities, achieving complete dye removal within 24 h. When combined with the biofilm form and the Ag/AgCl/Fe₃O₄ nanocomposite, the decolorization process was significantly accelerated, reducing the treatment time to 12 h. This improvement was validated through UV–Vis spectroscopy, which indicated a substantial reduction in dye concentration. The integration of bacterial consortia with nanocomposite technology is a highly effective, cost-efficient, and environmentally compatible approach to addressing textile dye contamination in wastewater. The use of biofilms and immobilization strategies not only improves the operational stability of the bacterial strains but also enhances their reusability, making this approach a viable option for large-scale industrial applications. Biotoxicity assessments showed that the decolorization effluent was significantly less toxic than the untreated dye mixture. Brine shrimp assays confirmed the reduced toxicity of the decolorized products, highlighting their safer environmental discharge profile.

Electronic Supplementary Material

Below is the link to the electronic supplementary material.

Author contributions

Fatemeh Heydari: Research & Investigation—Software and Simulation; Fereshteh Jookar Kashi: Idea & Conceptualization—Data Curation—Research & Investigation—Analysis—Software and Simulation—Funding Acquisition—Methodology—Project Administration—Supervision—Verification—Writing- Revise & Editing.

Funding

This research received no specific grant from public, commercial, or not-for-profit funding agencies.

Data availability

The data analyzed in this study is obtainable from the corresponding author upon reasonable request.

Declarations

Competing interests

The authors declare no competing interests.