Sample collection and bacterial isolation
Industrial effluent samples were collected in autoclaved screw-capped bottles from Kot Lakhpat industrial estate, Lahore, Pakistan. Physicochemical parameters such as pH, temperature, as well as color of the samples were also noted at the time of sample collection. The samples were serially diluted and plated on the L-agar plates. L-agar medium was prepared by dissolving tryptone (10 g), yeast extract (5 g), NaCl (5 g) and agar (15 g) in 1000 ml of distilled water. pH of the medium was adjusted to 7.
Evaluation of dye degrading potential
Dye degrading potential was determined by inoculating the bacterial isolate in 250 ml Erlenmeyer flask having 100 ml of mineral salt medium (MSM) whose composition is (g/l): (NH4)SO4, 0.28; MgSO4·7H2O, 0.04;NH4Cl, 0.23; KH2PO4, 0.067; FeCl3·6H2O, 0.005; CaCl2·2H2O, 0.022; yeast extract, 0.2; NaCl, 0.15; NaHCO3, 1.0 and 1 ml/l of a trace element solution containing (g/l): MnCl2·4H2O, 0.1;ZnSO4·7H2O, 0.01; CuSO4·5H2O, 0.392; NaB4O7·10 H2O, 0.177; CoCl2·6H2O, 0.248 and NiCl2·6H2O, 0.02 (Parshetti et al. 2006) with glucose and yeast extract (1% each) used as carbon and nitrogen source respectively. The medium was supplemented with dye (Synazol red 6HBN) at a concentration of 50 mg/l for 4 days of incubation at 37 °C. Synazol red 6HBN, Congo red, Methyl red, Phenol red, Brilliant black and Navy blue were purchased from Sigma-Aldrich. All other chemicals were of analytical grade purity.
Bacterial characterization
The bacterial morphological parameters and biochemical tests were performed according to protocols given in Cappucino and Sherman (2008). For 16S rRNA ribotyping, DNA was isolated (Masneuf-Pomarade et al. 2007) and 16S rRNA gene was amplified through PCR by using universal bacterial primers (Turner et al. 1999). PCR was performed according to Rehman et al. (2007) and PCR product was purified by Fermentas Gene Jet Gel Extraction kit (#K0691). The purified product was sequenced and submitted to GenBank for obtaining accession numbers. Phylogenetic analysis was done using MEGA7 (Kumar et al. 2016). Phylogenetic tree was constructed using neighbor joining method with 1000 replicates.
Determination of optimal growth conditions
The physical parameters which were favorable for the bacterial growth i.e., temperature and pH were analyzed by growing bacterium at different temperature and pH. For optimum temperature determination, bacterial isolate was grown in LB broth and incubated at different temperature i.e., 20, 30, 37 and 45 °C and for optimum pH, the bacterial isolate was grown in LB broth with pH values i.e., 5, 6, 7, 8, 9 and 10. After 24 h of incubation, absorbance was recorded at 600 nm by using spectrophotometer. In order to determine growth pattern of bacterial isolate, LB medium was inoculated with log phase grown bacterial culture (1 ml). Optical density was determined by spectrophotometer at 600 nm at the time of inoculation and then after regular interval of 4 h up to 28 h of growth at optimum temperature and pH.
Decolorization experiments
In order to optimize the decolorization conditions decolorization experiments were performed at various conditions i.e., temperature (20, 30, 37, 45 and 50 °C), pH (5, 6, 7, 8, 9, 10), incubation condition (static and shaking), carbon sources (saw dust, sugarcane baggase and wheat bran) and nitrogen source (yeast extract, beef extract and peptone) source, inoculum percentage (2%, 4%,6%, 8% and 10%) and dye concentration (3, 5, 7, 10, 20 and 50 mg/l). A volume of 100 ml of MSM was taken in 250 ml of Erlenmeyer flask which was then inoculated with 2% of bacterial suspension and incubated at respective condition. The stock solution of dye was added into the MSM to obtain a final dye concentration of 50 mg/l. Aliquot was taken out at 0 h and after 3 days in order to measure initial and final absorbance, respectively. The aliquot was centrifuged before measuring the optical density at 465 nm. Decolorization percentage of the sample was measured by using following formula. All the treatments and controls were carried out in triplicates.
$${\text{Decolorization }}\left( \% \right) = \frac{{{\text{Initial}}\,{\text{absorbance}} - {\text{Final absorbance}}}}{\text{Initial absorbance}} \times 100$$
Effect of decolorization on growth of bacterial isolate
In order to check the effect of decolorization on growth of bacterial isolate, the bacterium was grown in MSM containing dye concentration of 50 mg/l. The aliquot was obtained after every day up to 5 days and optical density was measured at 600 nm in order to find out growth ratio. Decolorization was also calculated by measuring the optical density of supernatant at 465 nm after centrifugation of the sample.
Decolorization of multiple dyes
The bacterial isolate was checked for its ability to decolorize a mixture of azo dyes i.e., Congo red, Methyl red, Phenol red, Brilliant black and Navy blue. The initial concentration of each dye was maintained at 50 mg/l. The optical density of centrifuged sample (supernatant) was taken and finally percent decolorization was calculated by using above mentioned formula (Kalyani et al. 2008).
Analysis of dye degraded products
HPLC, TLC and FTIR
The analysis of dye degraded products was done by thin layer chromatography (TLC), high performance liquid chromatography (HPLC) and Fourier transform infrared spectroscopy (FTIR). The metabolites were extracted from dye degraded sample (100 ml, 5 days) by mixing it with an equal volume of ethyl acetate. The extracts were then dried on anhydrous Na2SO4 and evaporated in rotary evaporator. The dried powder thus obtained was dissolved in HPLC grade methanol and used for analysis.
TLC was performed to analyze the degraded products on silica gel using mobile phase solvent system n-propanol, methanol, ethyl acetate, water and glacial acetic acid in the ratio 3:2:2:1:0.5 (Kalyani et al. 2008) and the results were visualized under UV illuminator at 254 and 366 nm. HPLC was performed at (Waters model no 2690) C18 column having symmetry 250 × 4.6 mm using methanol as mobile phase with a flow rate of 1.0 ml/min for 15 min and UV detector at 254 nm (Telke et al. 2009).
FTIR (Bruker, alpha-P) was performed to observe the change in structure of dye before and after decolorization. The FTIR analysis was done in the mid IRF region of 400–4000/cm. Prior to analysis sample was mixed with pure KBr in the ratio of 5:95 and pellet were then fixed in the holder for analysis (Saratale et al. 2009b).
Gas chromatography mass spectrometry
The collected samples were centrifuged for 10 min at 4 °C and the supernatant collected were extracted thrice with an equal volume of ethyl acetate, dried with Na2SO4 and further concentrated in the rotatory evaporator. GC–MS analysis of metabolites was carried out using gas chromatograph system (GCMS-QP2010 Ultra, Shimadzu) equipped with capillary column (DB-5 ms). The samples were diluted 1:100 and injected 1.0 µl sample into GC–MS in split mode with injector temperature of 290 °C. Helium was used as a carrier gas with flow rate of 1.02 ml/min under 54.9 kPa inlet pressure. The column temperature was set to 50 °C and hold for 1 min with subsequent increase to 280 °C with ramp rate of 30 °C/min without holding and finally to 310 °C with ramp rate of 15 °C/min by holding it for 8 min. The peaks were identified by comparing with NIST27.LIB mass spectra library of GC–MS database.
Effect of decolorized dye wastewater on microbial growth
The extent of bacterially treated wastewater effect was determined on some useful micro-flora including Bacillus megatarium (z-28), Bacillus cereus (T358-2) and Bacillus subtilis (z-66) according to Mali et al. (2000). The plates (L-agar) were swabbed by bacteria; a 2 mm well was made and filled with decolorized dye wastewater. The plate’s incubation was done at 37 °C for 24 h and the zone surrounded the well indicating the toxicity index of degraded dye sample.