Current World Environment

Materials and Methods

Sample collection, dyes, and chemicals

The tunnel developed by the bark borer insect (Indarbela tetraonis) in the trunk of the Peltophorum pterocarpum plant was used as a sample for the isolation of microorganisms for the decolorization and degradation of reactive red 120 (RR120) dyes. The samples were collected in a sterile zigzag polybag. The geographical coordinates for the sampling site were latitude (22.12821°) and longitude (82.13816°). RR120, phenol red, acid blue 113, Congo red, and Orange II, dyes were purchased from Sigma Aldrich. Bushnell Hass broth medium (BHM) was prepared as follows: (g/L^-1) FeCl₃ 0.05, CaCl₂ 0.02, MgSO₄ 0.2, and NH₄NO₃ 1.0, K₂HPO₄ 1.0, supplemented with glucose (0.4% w/v), 1% RR120 dye and yeast extract (0.2% w/v) media pH 7.0¹³.

Isolation, screening, and characterization of RR120 dye-degrading bacteria

A total of 3 (WR-1, WR-2, and WR-3) bacteria that break down reactive red 120 dyes were isolated from the bark borer tunnel (Figure 1A). A 100 ml flask containing 40 ml of BHM broth medium with 100 mg/L RR 120 dye, was filled with 5 g of the sample. The flask was then shaken at 120 rpm for 3 days at 35°C. Static anaerobic environments were maintained for three days at 35°C after 3 g of sample was added to 30 ml of BHM broth medium. Additionally, the decolorized samples (Figure 1b) were serially diluted, and plating was performed using dilutions of 10^–6 and 10^–7 on BHM agar supplemented with 100 mg/L Reactive Red 120. The isolated bacterial cultures were screened based on the percentage of decolorization. All isolated cultures were screened based on degradation efficiency, for which WR-2 was selected (Table 1). By staining and biochemical tests, the culture and colony morphology of the WR-2 isolates were identified. The genomic DNA of the positively isolated WR-2 was extracted for molecular characterization, and the 16F27 (5'-CCA GAG TTT GAT CMT GGC TCA G-3') and 16R1492 (5'-TAC GGY TAC CTT GTT ACG ACT T-3') universal primers were utilized for analyzing the 16S rRNA gene nucleotide sequence. An automated DNA sequence (ABI3730XL) was used to sequence the products. The EzBioCloud database was used for identification after the Lasergene package was used for assembly.

Table 1: Decolorization percentage of dye RR120 by the isolated bacterial cultures

Figure 1: a) Sampling site for isolation of bacteria, b) RR120 dye decolorized media by WR-2 (triplicate set) Click here to view Figure

Decolorization studies

BHM-containing media was added as a supplement. A screw cap tube containing 30 ml of BHM supplemented with 1 ml culture solution of bacteria grown throughout the night was added for inoculation. Medium without dye solution was used as the blank, and the dye-containing uninoculated media was used as the control. For 48–72 hours, at 37°C, the tubes were incubated. At 12-hour intervals during the incubation period, 5 ml of culture media was removed, and the bacterial cell mass was separatedafter centrifugation of mixture at 10,000 rpm for 10 minutes at 4°C. The absorbance of the culture supernatants at the RR120 dye maximum absorption wavelength (524 nm) was measured using UV-Vis spectroscopy to assess the decolorization potential. The formula was used to determine the percentage of decolorization, which was used to express the decolorizing efficiency¹⁴.

Microbial inoculums preparation

BHM broth was used to cultivate the isolates. Following the inoculation, the 30 ml screw-cap tubeswere placed at 35 °C in a static incubator until they reached their log phase, at which point they were used as inoculums (1.5×108 cells/ml).

Effect of aeration, inoculum concentration, and incubation time

The decolorization of RR120 was investigated in both static and continuously shaking environments. In 100 ml flasks, 30 ml of BHM with RR 120 dye (100 mg/L^-1) was included for the decolorization study under continuous shaking conditions. Then inoculated with a bacterial strain and maintained at 35 °C for 12–72 hours while being shaken at 150 rpm. In 30 ml screw-capped tubes, the inoculation medium was taken out, and the plates were placed in an incubator under static conditions for 12–72 hours at 35°C. Decolorization was studied at different incubation times (12 to 72 hours). The experiments were carried out with the previously described inoculation mixture in a static culture environment. The effect of different inoculum concentrations on decolorization was assessed at different concentrations (0.3–3 ml) of BHM. The BHM was then incubated under ideal static culture conditions for 12 hours¹⁴.

Taguchi Experimental Design

The 5 variables pH range 4-8, temperature 25°C- 45°C at 5°C intervals, 0.1, 0.2, 0.3, 0.4, and 0.5 mg/L RR120 dye Con; starch, lactose, sucrose, glucose, and fructose carbon sources (0.4% w/v); urea, ammonium sulfate, peptone, ammonium nitrate, and yeast nitrogen sources (0.2% w/v) were mutually exclusive and were used in the Taguchi design of the experiments. RR120 dye (100 mg/L^-1) was added to the BHM media. A suitable range of parameter alterations is included due to the selection of the 5 parameter levels. Tests were set up using the L₂₅ orthogonal array of TOM after the parameters that were effective at 5 different levels were selected. As previously mentioned, performing a large number of experiments is necessary for the full factorial design. When more factors are involved, the process becomes more complicated andlengthy. Taguchi recommended an orthogonal array technique, a particularly constructed method that reduces the number of tests needed to observe the full parameter space, as a solution to this problem. Thus, Taguchi suggested measuring performance traits that vary from the desired desired outcome using the loss function. The signal-to-noise (S/N) ratio is then obtained from the outcome of this loss function. To analyze the S/N ratio,three performance category parameters are usually used. The three categories are smaller-the-better, larger-the-better, and nominal-the-best¹⁵.

Immobilization of Alcaligenes faecalis subsp. phenolicus

A. faecalis subsp. phenolicus was cultured in LB media for 24 h at 35 °C and pH 7 before being immobilized. The cell pellets were extracted by centrifuging them for 10 minutes at 4°C at 5000 rpm. They were then washed with phosphate buffer solution (50 mM, pH 7.0) and suspended. The obtained cell suspension was mixed with sodium alginate (3%, w/v) and PVA (5%, w/v) in an even mixture. Using a syringe, the mixture was dripped dropwise into CaCl₂ (3%, w/v). As soon as SV-PVA cells were dripped into CaCl₂ (3%, w/v), beads (0.5 mm, 10 beads) were formed, which were kept in a shaker for 30 minutes. After restoring the cells for 48 hours in heterotrophic media, the produced beads were sterilized with distilled water and then kept in a CaCl₂ (3%, w/v) solution at 4^oC for further use. Bacteria-free beads were made for the control experiments^{16, 17}.

Batch-continuous decolorization

The studies were carried out in 30 ml screw-cap tubes that had 30 ml of RR120 dye, which included A. faecalis subsp. phenolicus immobilized 10 beads (0.5 mm size) and BHM broth medium. Batch decolorization tests were performed under static anaerobic conditions, 100 mg l^-1 dye with pH 7 and 35°C temperature . to evaluate the impact of operating and environmental factors on the efficiency of dye removal¹⁸. After 12 hours, the beads were transferred to a fresh medium so that nutrients continued to be provided, and batch decolorization of the dye continued to be performed.

Evaluation of azo dye and azo dye mixture decolorization by A. faecalis subsp. phenolicus

The A. faecalis subsp. phenolicus strain was used for this purpose, and each strain was inoculated individually in BHM broth medium before being incubated at 35-37°C. A. faecalis strains were utilized in studies to lessen azo dyes and mixed dyes (100 mg/l) from BHM media. Additionally, the percentage of dye decolorization at different incubation times was measured for the medium. Using a UV- 1900 UV?Vis spectrophotometer, the absorbance of the culture supernatants was measured at the maximum absorption wavelength of the azo dye mixture (525.6 nm), RR120 (524 nm), congo red (500 nm), phenol red (550 nm), orange II (484 nm) and acid blue 113 (570 nm)¹⁹.

Fabric decolorization

Fabric purchased from the local market in Bilaspur, Chhattisgarh, consisted of two sets of 100% cotton fabric (red and blue) cut into small, uniform pieces. The cloth pieces were properly packaged and autoclave-sterilized. The aseptic conditions of sterilized BHM broth media (30 ml) were used to separate tubes containing pieces of red and blue sterilized cloth. An overall quantity of 0.3 milliliters of inoculum was transferred to the test sample tubes, while the control medium remained uncultured. Anaerobic conditions were maintained for 2 days while all tubes were cultured at 35–37 °C²⁰.

Impact of heavy metals on the RR120 decolorizing potential of A. faecalis subsp. phenolicus

Cu, Pd, Zn, Ni, and Cd metal ions were used in a study to determine the efficacy of heavy metal tolerance. Separate additions of the heavy metals ranging from 1 to 6 mg/l were made to the dye decolorization reaction mixtures to examine the impact of ions from heavy metals on the biodegradation ability of Reactive Red 120. Additionally, all experiment was run in triplicate, and the previously indicated parameters were used when determining the decolorization rate. The dye decolorization rate measured in the lack of a bacterial strain was used as a control^21,22.

Enzyme assay

A. faecalis subsp. phenolicus strain was grown on LB broth media. For 24 hours, the culture tubes were incubated at 35 °C. After that, they were harvested by centrifuging the cells for 15 minutes at 4°C and 10000 rpm and then suspending them in pH 7.4 50 mM phosphate buffer. After centrifugation of homogenate for 15 minutes at 10,000 rpm, the supernatant was used as a source of crude enzymes²³. Biotic and abiotic cultures were used as native and positive controls, respectively ²⁴. The presence of tyrosinase, laccase, and manganese peroxidase in the cell-free extract was tested. 0.01% guaiacol in a 2 ml reaction mixture sample was mixed with 0.1 M phosphate buffer (pH 7.4) for laccase screening. Reactions between the released laccases and guaiacol resulted in a reddish-brown colour²⁵. The color change in the tubes containing 0.5 ml of thereaction solution with 0.5 ml of sodium tartrate buffer, 0.5 ml of guaiacol, and 10 mM H₂O₂ indicated the presence of manganese peroxidase. When the two substances and secreted manganese peroxidase react, a brownish-red color appears. The activity of tyrosinase was measured in a pH 7.4 0.1 M phosphate buffer with 2 ml of a reaction mixture containing 0.01% catechol. All enzyme screenings were performed at 30°C using reference blanks that lacked any component other than the enzyme. The enzyme screening test was carried out three times ²⁶.

EPS-producing strain study

To create the EPS, the strain was introduced into a commercial MRS medium at 37°C for 48 hours. After adding twice as much chilled ethanol and centrifuging it for 24 to 48 hours while it was chilled at 4°C, the precipitates were obtained^{27, 28}.

PGPR-producing strain study

NH₃ and indole acetic acid (IAA) production conformation tests have been studied as PGPR trait indices. The IAA generation of the bacterial strains was determined using the Gordon and Weber (1951) method²⁹. The IAA precursor L tryptophan was added to the LB broth and the A. faecalis sub sp. phenolicus strain was then added. IAA content and supernatant were measured after 48 hours of incubation. 1 ml of Salkowski reagent was introduced into the supernatants, which were then left at room temperature for 30 minutes to quantify IAA. The intensity of the generated pink color was checked³⁰. Rhizobacteria isolated from A. faecalis subsp. phenolicus were evaluated for the production of hydrogen cyanide (HCN)

The culture was grown on Kings medium broth supplemented with 4.4 g/L glycine, further, the culture tubes were incubated at 35°C for four days. The formation of HCN is indicated through the development of an orange or red color. Peptone broth medium was prepared for NH₃ production. To produce ammonia, this strain of rhizobacterium was cultivated in peptone broth and incubated for 48 to 72 hours at 35°C. After adding 0.5 mL of Nessler's reagent to the bacterial suspension following incubation. A color change from brown to yellow signifies the generation of ammonia³¹.

Decolorization and degradation analysis

FTIR, GCMS, and UV-visible spectroscopy studies were conducted on the Reactive Red 120 azo dye model to confirm the decolorization and degradation of the A. faecalis subsp. phenolicus strain. Bacterial strains were cultured in BHM growth media supplemented with 50 mg/L RR120 dye for 12 hours at 35–37 °C. Control and test samples were analyzed following centrifuge at 10,000 × g for 10 minutes at 4°C to study the UV?Vis, GC?MS, and FTIR results. According to Agrawal et al, the peak variations within the treated and control dyes were measured utilizing a UV-Vis spectrophotometer³². Based on earlier research, the decolorized broth was extracted two times with ethyl acetate in double quantities. To examine the composition alterations within the molecules of the dye found in the effluents when the dye biodegradation procedure, 16-scan speed FTIR dye and extracted metabolite spectra in the 400–4000 cm–1 mid-IR range were obtained. After diluting the sample residue with 1 ml of HPLC-grade methanol, GC-MS evaluation was performed immediately. Under oven conditions, the injector temperature in the GC was maintained at 300°C with split-way oven temperature and hold time (80°C-2min, 100°C-2min, 210°C-5min, 230°C-4min, 250°C-2min, 300°C-10min). A solvent delay of 3 minutes was selected, and helium was utilized as the carrier gas. The MS was run in total ion current (TIC) mode, scanning at 70 electron volts (eV) between 50 and 450 m/z. By comparing the mass spectra in the NIST library database, the dye decolorization metabolites were identified³³.

Results

Identification of selected isolate

The Bark borer, Indarbela tetraonis, of the Guru Ghasidas Vishwavidyalaya Bilaspur, campus contained the bacterial strain WR-2, which significantly degraded RR120 dye after 12 h. According to the result of the biochemical test of selected WR-2 strains, catalase, methyl red, citrate utilization, and mobility tests were found positive, and urease, indole, starch and gelatin hydrolysis, mannitol salt agar, phenylalanine deamination, gas, and H2S production test were found negative. The 16s RNA sequencing results showed that strain WR-2 was linked closely to Alcaligenes faecalis sub sp. phenolicus DSM 16503(T) accession number AUBT01000026 at 98.84%. The 16S rRNA gene sequences of the WR-2 strain from the NCBI and Mega 11 databases were used to construct a phylogenetic tree (Figure 2). The sequence has been uploaded to Gen Bank (MTCC) with accession number 13437.

Optimization of the impact of agitation Shaking and Static, time, and inoculum size in RR120 decolorization

Shaking and Static conditions for cell growth are shown in Figure 3a. At the cell growth stage, oxygen has an important effect on physiological properties. Oxygen can either promote or prevent azo dyes from being broken and degraded by microbes. Under static conditions, the A. faecalis subsp. phenolicus strain decolored RR120 96%; however, under shaking conditions, only 32.47% decolorization was observed (Figure 3b). Under anaerobic and static conditions, A. faecalis subsp. phenolicus could completely decolorize RR 120 (100 mg/l) after 12 hours of incubation (Figure 3b). A. faecalis subsp. phenolicus was shown to decolorize RR120 dye in 0.3–3 ml of inoculums (Figure 3c). The decolorization rate increased significantly with a subsequent increase in inoculum size above the optimal value.

Figure 3: Impact of a) growth, b) static and shaking conditions; c) inoculum size on RR120 decolorization by A. faecalis subsp. phenolicus Click here to view Figure

Taguchi analysis

Table 2: L₂₅ Orthogonal observation using the Taguchi method for RR120 dye degradation

Table 3: Response observation for means value and signal-to-noise ratio

Figure 4: a) Main effects for means of means b) Main effects for SN ratios means of SN ratios Click here to view Figure

Repeated-batch decolorization

Complete decolorization was observed using immobilized bacterial beads for up to 25 cycles in anaerobic environments. After 5 cycles, the rate of decolorization decreased significantly; however, with the bacterial beads immobilized in this study, more than 95% decolorization was still observed in subsequent cycles (Table 4). For RR120 decolorization, the immobilized bacteria-mediator beads demonstrated excellent reusability.

Table 4: Decolorization percent of RR120 by using immobilized beads

Decolorization of azo dye and dye mixtures

The bacterial strain was subsequently analyzed using a BHM medium supplemented with 300 mg/l concentrations of the corresponding dyes for the decolorization of AB 113, orange II, congo red, RR120, and phenol red individual dye and dye mixtures. For all dyes, there were differences in the rate of decolorization. It decolored 76.51% of AB 113, 96.8% of orange II, 98.47% of congo red, 98.3% of RR120, 97.92% of phenol red individual dyes, and 94.72% of the dye mixture at the 12-hour mark of the incubation period. It decolorized 99.1% of AB 113 after 24 hours of incubation. The nature and position of the substituent in aromatic rings, as well as the reaction between the azo link and various dyes, are the main factors influencing the variations in the dye decolorization pattern. These variations also affect structure, complexity, and specificity, according to reports.

Enzyme assay

The difference between the production of enzymes found in the control and the cells produced after decolorization is indicated by the results. It is likely that biotransformation enzymes, such as laccase and MnP, are primarily responsible for the decolorization of cells. For every microbe, there may be differences in the appropriate contributions of MnP and laccase to the decolorization of dyes. According to previous studies, a significant enhancement in the activity of these enzymes during decolorization suggests that these enzymes are involved in the dye breakdown process.

PGPR activity

The production of HCN, IAA, and NH3 was found to have good potential in the A. faecalis subsp. phenolicus strain. After 48 hours of incubation under static conditions, the cells were observed.

EPS production

The extracellular polysaccharide that was isolated for this investigation, however, is a white powder. The pale yellow color of the powder could have resulted from bacteria. No powder was obtained in the control tubes because of the EPS production capacity of the Alcaligenes faecalis subsp. phenolicus strain was compromised.

Fabric decolorization

The fabric was separated from each tube, then washed 3 times with phosphate buffer, and dried at room temperature. Comparative observation and Raman Microscopic (Figure 5 a) and b) were performed on the color of the control fabrics and bacteria-treated fabrics. Compared with that of the control, the color of the bacterial-treated fabric became colorless. The fabric decolorization test showed the capability of bacteria to remove dye from the colored fabric.

Figure 5: Fabric decolorization observation (A), (B) Microscopic images Click here to view Figure

Metal tolerance activity

According to the amount of time needed for the RR120 azo dye to completely decolorize, the impacts of heavy metal ions on the strain's ability to decolorize dye were studied. Figure 6 shows the results obtained from experiments carried out with Cu, Pd, Zn, Ni, and Cd heavy metal ions present at various concentrations. The A. faecalis subsp. phenolicus strain required 12 hours to completely decolorize Reactive Red 120 when no heavy metal ions were present. Remarkably, in the presence of metal tolerance, the time needed for the rate of 100% decolorization was reduced by around 72 hours. When the amount of metal ions, viz. Zn, Cu, Pd, and Ni, increased from 2% to 6%, there was a minor decrease in the amount of 72 h needed for RR120 to decolorize 100% of the media sample. >72 h required for 100% dye decolorization at a concentration of 1-6% for the metal ion Ni. Textile effluents with heavy metal ions may have issues with low biodegradability, extending the time required for biological treatment.

Figure 6: Metal ion effects on the percentage of dye degradation. Click here to view Figure

UV, FTIR, GCMS analysis

The most common and basic technique for analyzing dye decolorization is UV-Vis spectroscopy. Figure 7 clearly shows how the decolorization of the RR120 azo dye results in the elimination of the sharp peak at the maximum wavelength of RR120 (?max 524 nm), showing that the dye within the medium was destroyed by the bacteria after a 12-hour incubation period. Additionally, FTIR is often used as a suitable technique to examine the degradation of dyes. FTIR is a useful analytical technique in dye degradation research because it can be used to determine the type and degree of interactions that take place inside azo dyes with many functional groups which are degraded by bacteria. Therefore, the biodegradation method was studied using the mid-IR 400–4000 cm-1 range of FT-IR spectroscopy in addition to the previously discussed analytical methods. After mixing the samples with spectroscopically pure KBr, the analyses were completed. The presence of the dye compound is indicated by a medium peak in the control RR120 dye spectrum that revealed an O-H stretch of the phenolic group at 1381.38 cm^?1 and a C?=?N stretch of the amine at 1130.61 cm^-1 and 1028.09 cm^-1.When the FTIR spectrum of RR120 was compared to that of its degraded metabolites after 24 hours, it was evident that A. faecalis subsp. phenolicus biodegraded the dye. Additionally, the FTIR spectra of the extracted metabolites revealed a moderate peak at 3353.43 cm^-1 corresponding to the formation of aliphatic primary amines through N–H stretching, with a strong peak at 1555.37 cm^-1 corresponding to the N–O stretch and the formation of nitro compounds. Halo compound C-I stretching is indicated by the medium peak at 534.52 cm^-1. The results are displayed in Figure 8A and B. In RR120 dye, ten peaks were found during GC-MS analysis. RR120 dye components were identified Dodecane, 2, 6, 11-trimethyl-$$ 2, 6, 11-Trimethyldodecan, Tetrapentacontane, Tetrapentacontane, Bis (2-ethylhexyl) phthalate $$ Phthalic acid, and Hexatriacontane $$ n-Hexatriacontane. The broken-down metabolites of Reactive Red 120 were identified by GC-MS analysis (Figure 9, Table 5) and organic solvent extraction as pentanoic as acid (molecular mass 116, m/z 116, retention time 3.297, area 24.38%), pentanoic acid (molecular mass 116, m/z 116, retention time 5.253, area 0.35%), 2-piperidinone (molecular mass 99, m/z 99, retention time 13.617, area 39.28%), methyl tetradecanoate (molecular mass 242, m/z 242, retention time 23.934, area 0.27%), 1,4-diazabicyclo[4.3.0]nonan-2,5-dione (molecular mass 168, m/z 168, retention time 24.307, area 3.35%), cyclo(L-prolyl-L-valine) (molecular mass 196, m/z 196, retention time 25.490, area 12.77%), cyclo(L-prolyl-L-valine) (molecular mass 196, m/z 196, retention time 25.981, area 1.94%), and pyrroline This set off a series of events that eventually produced a stable intermediate.

Figure 7: UV-spectroscopy analysis Click here to view Figure

Figure 8: FTIR analysis graph of (A) RR120 dye and (B) RR120 dye degraded metabolites Click here to view Figure

Figure 9: GC-MS analysis graph of degraded RR120 metabolites Click here to view Figure

Table 5: GCMS identified compounds and degraded metabolites of RR120 dye

Discussion

According to a study by Rehaman et al, 74% and 85% of the PGPR Azospirillum sp. and Pseudomonas sp. decolorized reactive blue dye at 37°C, respectively, within 20 days³⁵. Maniyam et al noted that the decolorization effectiveness of the methyl red dye was greater in a static situation than under shaking conditions for two Rhodococcus strains UCC 0016 and UCC 0008³⁶. In this study, the PGPR A. faecalis sub sp. phenolicus was able to decolorize RR120 (0.1 mg/l), which reached 99% at pH 7, 35°C, 1% inoculum, and under static conditions within 12 h. Among the 8 azo dyes, namely, reactive blue R, Janus Green B, ponceau S, cibacron brilliant Red, Evans Blue, Alura Red AC, and brilliant green Bacillus subtilis was determined to be the most effective bacterium. The decolorization rate ranged between 71.8% and 100%³⁷. A. faecalis subsp. phenolicus was able to degrade many azo dyes (76.51% of AB 113, 96.8% of orange II, 98.47% of congo red, 98.3% of RR120, and 97.92% of phenol red individual dyes and 94.72% of dye mixture), according to this work. According to the results, using a carbon source increased Enterococcus faecium's methyl orange decolorization by 100% and in Bacillus cereus by 75%³⁸. According to this study, using a fructose and peptone carbon nitrogen source and 0.1% dye contraction increased the decolorization of RR120 by A. faecalis subsp. phenolicus 100%.To decolorize Congo red dye Goswami et al, combined ozonation with a packed bed bioreactor that contained biochar immobilized with Providencia stuartii³⁹. As a pre-treatment, the biological method removed dye with an efficiency of 92%. The complete decolorization of RR120 by A. faecalis subsp. phenolicus-immobilized cells in a batch continuous reactor were used in this study. Bacillus megaterium KY848339.1 was capable of decolorizing the acid red 337 azo dye at an optimal pH 7 and 91% efficiency at a primary concentration of 500 mg/l in less than a day. Dye biodegradation is influenced by effluent pH. Bacillus sp. removes the azo dyes Navy blue and methyl red at different pH values (5–8)⁴⁰. The degradation efficiency of Bacillus sp. strain CH12, when various carbon sources were added, was greater than that of the carbon-free culture (27–51%), with decreasing efficiency of 95% to 100%. In contrast to inorganic sources, which produced the maximum degradation efficiency, the inclusion of peptone and yeast extract as a source of nitrogen produced the highest degradation efficiency (>90%) in the same study⁴¹. Parma and Shukla investigated how various dye concentrations affected the way Staphylococcus hominis subsp. DSM 20328 degraded the C.I. Reactive Blue 4, an anthraquinone-based dye. They studied an increase in the dye's quantity followed by a reduction in the degradation efficiency of dyes⁴². Arsenic, nickel, copper, and zinc salts were tolerated by the moderately halotolerant strain of Klebsiella at concentrations of 1.25 mM, 1.5 mM, 3 mM, and 5 mM, respectively, because they degraded Congo red and Acid Orange 7⁴³. In this study, the A. faecalis subsp. phenolicus strain decolorized RR120 when heavy metals were present (1% Ni, Pd, Cu, Cd, and Zn).

Conclusion

Reactive red 120 dye was decoloured in simulated BHM using heavy metal-tolerant and EPS-producing A. faecalis subsp. phenolicus strains under various physicochemical and operational conditions. Comparing Taguchi's method of parameter design to full factorial analysis, the latter requires fewer experiments and obtains comparable results. The dye concentration, pH, carbon-nitrogen sources, and temperature all affected decolorization. A temperature of 35°C, pH of 7, dye concentration of 0.1 mg/l, carbon source of fructose, and nitrogen source of peptone, a duration of 12 hours, static conditions, and 0.3 ml of inoculums were the most suitable parameters for Reactive red 120 dye removal. The immobilized beads were found to be stable for 12 hours in repeated batch cultivation for RR120 degradation when A. faecalis subsp. phenolicus was used continuously for this purpose. The results of this investigation demonstrated the great decolorization efficiency of this A. faecalis subsp. phenolicus bacteria strain. This strain decolored 76.51% of AB 113, 96.8% of orange II, 98.47% of congo red, 98.3% of RR120, 97.92% of phenol red individual dyes, and 94.72% of the dye mixture at the 12-hour. Aliphatic primary amines, nitro compounds halo compounds were detected by FTIR analysis in the isolated metabolites that were produced during dye decolorization. GC-MS analysis was used to identify low molecular weight substances. The significant removal of the color rate of the strain of bacteria in natural environments suggests that it has the potential to be both economically and environmentally beneficial when used in the microbial treatment of dye-containing effluents.

Acknowledgment

The authors are thankful to the National Centre for Microbial Resource, Pune, Maharashtra, and the Central Instrumentation Lab, Central University of Punjab, Bhatinda, for GC-MS analysis.

Funding sources

The author(s) received no financial support for the research, authorship, and/or publication of this article

Conflict of interest

The authors do not have any conflict of interest.

Data Availability Statement

All dataset incorporated in the manuscript are produced by the authors

Ethics Approval Statement:

This research did not involve human participants, animal subjects, or any material that requires ethical approval

Authors contribution statement

Kusumlata; writing—original data preparation, Rajat Pratap Singh; review, Ashish Kumar; Data analysis, review, editing and visualization.

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