Hussain et al. [19] provided a foundation for research on Novacron Red SB decolorization, but our study advances research on it in several ways. We used dye concentrations that are typical of actual textile effluent. We also isolated bacteria with different decolorization profiles. We performed a more precise pH optimization (4-9 at 0.5 intervals) to determine the true optimum. Interestingly, we found ammonium acetate to be the most effective co-substrate, which differs from the earlier finding of peptone. We also used statistical analysis to validate all comparisons. Finally, we tested our optimized conditions in real textile effluent, which was not performed previously. Together, these improvements offer a more comprehensive foundation for using these bacteria in wastewater treatment.

Effluent samples were collected from different locations of the drain of Clifton Apparels Ltd. (Composite), 179, Baizid Bostami Road, Nasirabad Industrial Area, Chattogram, Bangladesh, in sterile glass bottles. To maintain sample integrity, each sample was placed in an insulated box with frozen refrigerant packs immediately after collection and preserved at 4°C upon arrival in the lab.

Bacterial strains were isolated by repeatedly streaking the collected samples onto nutrient agar (Hi-Media, India) plates. Distinct colonies were selected for isolation based on colony morphology. Each distinct colony was carefully picked up and transferred onto a nutrient agar slant. Slants containing different colonies were prepared, sealed in a polythene bag, and stored at 4°C for subsequent analysis. To maintain the isolates’ characteristics, subculturing was performed at 3-week intervals.

Bacterial isolates were inoculated into Minimal Salt Medium (MSM) supplemented with 0.005% Novacron Red SB dye [20]. For comparison purposes, a negative control was prepared by adding Novacron Red SB azo dye to minimal salt medium (MSM) without inoculating bacterial isolates. These experiments were carried out in the dark to eliminate any potential photodegradation of the dye. Both the tested and control samples were incubated at 37°C for 72 h under shaking conditions. After incubation, dye decolorization in tested samples was assessed visually by comparison with the negative control (Figure 1d). To reduce bias, two independent observers assessed decolorization, and only isolates showing clear color reduction were selected. The reduction in color intensity was considered as the ability of the strain to decolorize Novacron Red SB, which was preserved for further analysis.

We selected isolates showing significant dye decolorization for identification. Cultural properties, microscopic examinations and conventional biochemical tests i.e. motility test, Indole test, Voges-Proskauer test, methyl-red test, Simmons’ citrate test, nitrate reduction test, catalase test, starch hydrolysis test, gelatin hydrolysis test, oxidase test, urease test, triple sugar iron test, and hydrogen sulfide production test, were performed. All microscopic, physiological and biochemical observations were compared with standard descriptions from Bergey’s Manual of Systematic Bacteriology, 2^nd edition (2001-2012) [21] and the bacterial isolates were presumptively identified up to species level.

The bacterial isolates were cultured in MSM broth (pH 7) and incubated until mid-logarithmic phase. The inocula were adjusted to optical densities at 625 nm (OD₆₂₅) of 0.002 for Gram-positive strains and 0.008 for Gram-negative strains, as measured using a UV-Visible spectrophotometer (Shimadzu UV-VIS 1800, Japan). OD₆₂₅ was used as a relative turbidity measure; viable cell numbers were not independently calibrated.

We used these OD values consistently across all experiments. A 0.015% solution of Novacron Red SB (prepared in distilled water) was centrifuged at 6000 rpm for 10 min for the determination of the maximum wavelength (λ_max) of the dye to remove any undissolved dye particles or suspended impurities. The OD of the dye was measured in the range of 400-700 nm using a UV-Visible spectrophotometer (PG Instruments - UK, Model: T 80+ UV-VIS), and its maximum absorbance (λ_max) was determined at 510 nm. The dye solution of 0.0003-0.01% at 2-fold dilution was diluted in MSM. After centrifugation, the OD of each dilution was measured at 510 nm, which was identified as λ_max of the dye. Novacron Red SB decolorizing efficiency of the isolates was calculated from the values of OD at 510 nm and presented as percent (%) decolorization = (A_i - A_t) × 100/A_i. Here, A_i and A_t are the OD at initial and treated time, respectively.

To determine the optimal conditions for Novacron Red SB decolorization, various physicochemical parameters were studied. These physicochemical parameters are co-substrate, temperature, pH, time, and dye concentration. To identify the best-fitting parameters, we conducted several pilot experiments. All experiments were performed under shaking conditions at 120 rpm using a 5% (v/v) inoculum of a 24-hour-old bacterial culture. For example, 5 mL of culture was added to 95 mL MSM, and the same 5% (v/v) ratio was maintained for other working volumes. A control was run to examine any abiotic degradation. Each experiment was conducted in triplicate.

0.005% (50 mg/L) and 0.010% (100 mg/L) Novacron Red SB dye concentrations were applied to determine the condition that provided maximum decolorization. Other environmental factors were kept constant, i.e., the medium’s pH was set at 7 and the temperature at 37°C. We repeated the experiments at 24 h intervals to determine the maximum decolorization.

To assess the effect of carbon-rich co-substrates on bacterial decolorization, glucose, ammonium acetate and peptone at a specific concentration (4 g/L) were added separately in MSM medium with 0.005% (50 mg/L) dye concentration and 72 h incubation period. These co-substrates were selected based on their frequent use in the literature and the objective was to compare their effectiveness in supporting decolorization by the isolates. Controls (MSM medium with co-substrate but without dye) were used to zero the spectrophotometer before measuring each sample.

The medium was tested at pH 4-9 at 0.5-unit intervals, with a 0.005% dye concentration and 4 g/L co-substrate concentration (ammonium acetate) and a 72 h exposure time. We used controls (medium with dye, no bacteria) at each pH tested. We accounted for any absorbance reduction observed in the controls.

Different temperatures i.e. 25°C, 37°C and 45°C, were used to determine the optimum temperature and 0.005% dye concentration, 4 g/L co-substrate concentration (ammonium acetate), and pH 7 of the medium, and 72 h exposure time were applied.

Decolorization efficiency of the dye by bacterial isolates was examined to assess the potential of the optimized condition to work in real textile effluent under laboratory conditions. The experiment was conducted by adding 4 g/L ammonium acetate as a co-substrate to the effluent, adjusting the pH, sterilizing the effluent by filtration through a sterile 0.22 µm filter and then co-incubating with the bacterial isolates for an incubation time 72 h at 37°C. In contrast, another textile effluent sample was taken and sterilized without pH adjustment or the addition of any co-substrate. This sample was then incubated with bacterial isolates at 25°C (effluent temperature) for 72 h, which was considered an unoptimized condition. The dye decolorizing ability of the bacterial strains under optimized conditions was evaluated by measuring the absorbance reduction at 510 nm using spectrophotometry.

All experiments were performed with three independent biological replicates per treatment and time point (n = 3). Data are presented as mean values; error bars represent standard deviation (SD). Statistical analyses were performed using GraphPad Prism version 11. We inspected distribution and variance graphically and observed no obvious violations. For the time-course experiments, separate two-way ANOVAs were performed for each bacterial strain to evaluate the effects of conditions and incubation time (24, 48, and 72 h) and their interaction. The treatment factors were: pH (4.0-9.0) for the pH experiments; dye concentration (50 mg/L, 100 mg/L) for the dye experiments; temperature (25°C, 37°C, and 45°C) for the temperature experiments; co-substrates (glucose, peptone, ammonium acetate) for the co-substrate experiments. For the optimized vs. unoptimized comparison, the factors were condition (optimized vs. unoptimized) and bacterial strain (B. megaterium, K. aerogenes, and C. rathyi). For the pH data at 72 h, we also performed separate one-way ANOVAs for each strain to compare pH levels, followed by Tukey’s HSD test. Following a significant ANOVA result (p < 0.05), Tukey’s HSD test was used for all pairwise multiple comparisons. We set statistical significance at p < 0.05. Different letters above the bars denote statistically significant differences between experimental groups, while shared letters denote no statistically significant differences.

Initial analysis of the textile effluent samples showed a high load of pollution markers. The samples exhibited pH values ranging from mildly acidic to neutral. The effluent temperature measured around 25°C. Other physicochemical properties were recorded (Table 1).

This study utilized Novacron Red SB dye-containing effluent from Clifton Apparels Ltd. (Composite) of Chattogram city as a source of naturally occurring bacteria. The repeated streak plate method was applied until an identical single colony appeared. Five distinct bacterial strains were selected based on their colony properties.

We investigated the dye-decolorizing ability of the isolates in MSM. A 0.005% concentration of Novacron Red SB dye was added to the medium, with pH maintained at 7.2 (equivalent to the effluent pH). The inoculated medium was incubated for 72 h at 37°C, and the reduction in color intensity was observed by naked eye. To reduce bias, two independent observers assessed decolorization, and only isolates showing clear color reduction were selected. Among the 5 isolates, NRD I, NRD II, and NRD III exhibited clear signs of decolorization at 72 h and were selected for further investigation.

The microscopic, cultural, physiological and biochemical characteristics of the selected strains were observed (Table 2, Table 3 and Table 4) and compared with the standard descriptions of Bergey’s Manual of Systematic Bacteriology, 2nd edition (2001-2012) [21], and the organisms were presumptively identified as Bacillus megaterium (NRD I), Klebsiella aerogenes (NRD II), and Corynebacterium rathyi (NRD III). Figure 1 presents their microscopic views.

The decolorization of Novacron Red SB was investigated against a number of parameters e.g., dye concentration, time, co-substrate, pH and incubation temperature.

Figure 2 shows that decolorization efficiency was significantly higher at 0.005% than at 0.010% dye concentration after 72 h. However, post-hoc analysis (Two-way ANOVA followed by Tukey’s test) revealed that this concentration-dependent effect was significant only for B. megaterium and K. aerogenes (p < 0.05), and not for C. rathyi (p > 0.05).

In our study, B. megaterium consistently showed high decolorization across most conditions tested. All of them decolorized the 0.010% dye solution to a lesser extent. In this study, the highest dye decolorization was observed after 72 h.

Among the co-substrates tested for efficient dye decolorization, the type of co-substrate had a significant overall effect (F(2,72) = 360.1, p < 0.001, η² = 0.437). Post-hoc comparisonsshowed that ammonium acetate was a significantly better co-substrate than both glucose (p < 0.001) and peptone (p < 0.001) (Figure 3). There was no statistically significant difference between glucose and peptone.

The decolorization of Novacron Red SB dye by the strains was examined in the pH range of 4-9 with 0.5 interval (Figure 4). Maximum decolorization activity was recorded at pH values of 6.5-8.0 (all p-values < 0.001). Among different pH levels tested for efficient dye decolorization, pH had a significant overall effect for B. megaterium (F(10,66) = 776.2, p < 0.001), K. aerogenes (F(10,66) = 539.0, p < 0.001), and C. rathyi (F(10,66) = 448.3, p < 0.001) by two-way ANOVA, with significant time and pH × time effects also observed for all strains (all p < 0.001). As the pH × time interaction was significant, one-way ANOVA was performed at 72 h for each strain. Tukey’s HSD showed that B. megaterium peaked at pH 6.5 (80.0 ± 2.65%); F(10,22) = 403.8, p < 0.001. pH 6.5 was significantly higher than pH 4, 4.5, 5, 5.5, 6, 7, 7.5, 8, 8.5, and 9 (each comparison, p < 0.001). K. aerogenes peaked at pH 8.0 (76.0 ± 3.00%); F(10,22) = 345.7, p < 0.001. pH 8.0 was significantly higher than pH 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8.5, and 9 (each comparison, p < 0.001). Corynebacterium rathyi peaked at pH 7.0 (67.0 ± 2.65%); F(10,22) = 236.1, p < 0.001. pH 7.0 was significantly higher than pH 4, 4.5, 5, 5.5, 6, 6.5, 7.5, 8, 8.5, and 9 (each comparison, p < 0.001).

Decolorization increased with temperature from 25°C to 37°C (Figure 5). A further increase in temperature to 45°C significantly impaired bacterial decolorization capacity. The optimum temperature for decolorization of Novacron Red SB dye by the bacterial strains was 37°C, with 85%, 80%, and 71% decolorization by B. megaterium, K. aerogenes, and C. rathyi, respectively. A two-way ANOVA on decolorization percentages after 72 h with temperature and strain as factors showed a significant effect of temperature (F(2,18) = 619.1, p < 0.001, η² = 0.986) and strain (F(2,18) = 24.2, p < 0.001, η² = 0.729), with no significant temperature × strain interaction. Decolorization at 37°C (mean = 78.7%) was significantly higher than at both 25°C (mean = 33.7%) and 45°C (mean = 17.3%). Tukey’s HSD test revealed that decolorization at 37°C was significantly higher than at 25°C (p < 0.001) and 45°C (p < 0.001). Therefore, 37°C is the optimal temperature for decolorization.

To validate the optimized conditions in the textile effluent, the pH of the effluent sample was first adjusted to 7 as the optimum. The pH of 7.0 was selected for this experiment because the textile effluent used in this study had a natural pH of 7.2, making pH 7.0 environmentally relevant for the effluent matrix. However, future studies should optimize pH for each strain individually before scaling up. Then the effluent was sterilized to avoid the effect of microbial contaminants in the sample and co-incubated with the bacterial isolates for the optimized time and temperature, 72 h and 37°C, respectively. All of these optimum parameters were combined and dye decolorization was performed against two solutions. One was a dye solution of known composition and the other was textile dye effluent containing a complex composition. Figure 6 illustrates the decolorization efficiency against these two solutions by the bacteria. All bacteria showed better decolorization under optimized conditions than under unoptimized ones. Among these bacteria, B. megaterium showed high decolorization under the optimized conditions.

The maximum decolorization occurred at 37°C and near-neutral pH (7.0) following a 72-hour incubation period.

1. 16S rRNA sequencing was not performed for this study due to funding limitations. Future work should employ 16S sequencing to confirm species identity and explore genomic potential.

2. The “naked eye” observation was used only as a rapid, preliminary test to eliminate non-decolorizing isolates from the total isolated bacteria. We performed all subsequent quantitative experiments using spectrophotometric measurements at 510 nm.

3. Our study did not identify or quantify degradation products or metabolites, nor assess their ecotoxicity. Future studies should include enzyme activity assays, identification of metabolic intermediates, toxicity evaluation, and elucidation of the degradation pathway to provide a more comprehensive mechanistic understanding of the decolorization process.

4. Azo dye reduction can occur under aerobic conditions via alternative mechanisms (e.g., aerobic azoreductases, laccases, or non-enzymatic adsorption). All experiments were performed under aerobic shaking conditions, and the exact mechanism was not confirmed. Aerobic shaking conditions excluded obligate anaerobic bacteria. Consequently, the decolorization efficiencies reported in this study represent only the capacity of aerobic and facultative anaerobic isolates. Future studies could employ anaerobic enrichment strategies to capture obligate anaerobes and compare their dye-degrading capabilities under anoxic conditions.

5. The relatively coarse temperature gradient and the use of only two discrete dye concentrations limited our ability to determine the precise optimal temperature and concentration for maximal decolorization.

6. The textile effluent was sterilized before inoculation to eliminate native microbial populations that would normally interact with the introduced strains in a treatment plant. Sterilization does not simulate real treatment conditions and so non-sterile validation is needed before scale-up.

7. Salinity or different ion concentrations, heavy metal concentrations, and exact dye concentration in effluent were not analyzed in this study. Future studies should include comprehensive physicochemical characterization to better understand the factors influencing microbial community composition.