Biodegradation Studies on Reactive Textile Dyes using Indigenous Bacterial Isolates and their Application in Membrane Bioreactor

Abstract

Textile dying and manufacturing industries are polluting valuable water resources and treatment of textile dye containing effluent needs a lot of scientific scrutiny. Decolorization of dyes has received much attention due to their toxicity, carcinogenic and mutagenic behavior. Reactive textile dyes are among the most important and widely used group of synthetic dyes and recalcitrant xenobiotics. Conventional technologies are unsuccessful for efficient and complete removal of these compounds from contaminated environments. Effective and eco-friendly treatment of these dyes can be achieved by the process of bioremediation, of which bacterial mediated degradation, being fast and efficient, is very important. Therefore, the aim of this current research work was to evaluate the degradation of reactive textile dyes by bacterial strains isolated from waste disposal sites of local textile industries and the application in a membrane bioreactor for treatment of simulated textile wastewater. Initially, thirty indigenous bacterial strains were isolated from a soil sample collected from textile waste disposal site and screened for their decolorization potential against three representative dyes of reactive class, i.e. Reactive Blue 221 (RB 221), Reactive Yellow 145 (RY 145) and Reactive Red 195 (RR 195). Three bacterial strains, viz. Pseudomonas aueroginosa SAD1, Alcaligenes sp. SAD2 and Brevibacillus parabrevis SAD3, showing maximum decolorization activity against model dyes were selected and taxonomically characterized based on morphological, biochemical and 16S rRNA gene analysis. As a step forward, different nutritional and cultural parameters were optimized by an one factor at a time (OFAT) strategy in the batch experiments in order to get maximum decolorization activity for all three isolates. The factors selected for optimization included temperature, pH, inoculum size, dye concentration, NaCl concentration, nitrogen, carbon source and their concentration. As a result of optimization, best decolorization of selected dyes by selected bacterial strains was achieved in mineral salt media containing glucose and yeast extract as the most preferable carbon and nitrogen source, respectively. The mesophilic temperature range (30-370C) was suitable for maximum decolorization activity. The pH range 7-8 was observed to be suitable for decolorization and strains could also tolerate higher concentrations of dyes and NaCl. The optimization studies resulted in an increase in decolorization potential of bacterial isolates. Therefore, fractional factorial design was used to find out the best conditions for decolorization of dyes using a developed consortium. The optimum culture conditions for decolorization of RB 221 (200 mg/L) by consortium were found to be 300C, 20% inoculum size, pH 7, 10 ml/L of trace element solution, glucose concentration (carbon source, 2 g/L) and yeast extract (nitrogen source, 1 g/L) and 98.68% of the dye was decolorized in 48 hrs of incubation. However, almost complete decolorization (>99%) of RY 145 was achieved at 370C, 20% inoculum size, pH 9, 10 ml/L of trace element solution, glucose concentration (1 g/L) and yeast extract (0.5 g/L) within 72 hrs of incubation. On the other hand, optimal operational and xiinutritional conditions for maximum color removal of RR 195 (200 mg/L) were found to be 370C, 10% inoculum size, pH 7, 20 ml/L of trace element solution, glucose concentration (2 g/L) and yeast extract (0.5 g/L); and decolorization activity was reached to 96.87% at 48 hrs. The dye degradation profile of dyes was initially studied by FTIR. The mid-IR fingerprinting region (400-4000 cm -1 ) of FTIR produced characteristic peaks of different overlapping functional groups of the parent dye molecule. The FTIR spectrum of a control dye and the extracted metabolites of complete decolorization by consortium was compared and significant difference in peak position, their intensity and number was observed. In treated samples, along with change in intensity, formation of new peaks along with disappearance of some peaks was observed, which provide a clue about the biodegradation of the dye into subsequent compounds. The peak representing the azo linkage (1658.58 cm -1 , 1597.12 cm -1 ) was completely absent in the treated sample and disappearance of different characteristics of aromatic systems and cleavage of azo bond (-N=N-) clarified efficient degradation of dyes. The products formed as a result of degradation of dyes were analyzed by GC-MS and spectroscopy analysis confirmed molecular degradation and the formation of secondary metabolites. The GC-MS spectra indicated the conversion of RB 221 into dimethyl phthalate at retention time of 8.576 with m/z 194 and benzoic acid methyl ester at retention time of 2.964 having m/z 136. In the case of RY 145, the formation of low molecular weight aromatic compounds during degradation of RY 145 by the consortium was observed. The metabolites were identified as phthalic anhydride at retention time of 6.53 with m/z 148, benzoic acid methyl ester with m/z 136 at retention time 4.06 and aniline (m/z 93) at 2.84 retention time. The degradation products for RR 195 were identified as benzoic acid methyl ester and 2-Napthol with base peak at m/z 136 and m/z 144, respectively. Along with that, methyl phthalate and aniline were also identified showing their corresponding peaks at m/z 163 and 93, respectively. The toxicity assessment of dyes and metabolites generated after the degradation process is one of the prime responsibilities in view of environmental safety and remediation. Toxicity was calculated by observing the mortality percentage of Artemia salina. Lower mortality rates were observed with treated samples in comparison to the parent dye solutions. Among treated samples, the lowest toxicity (0-10%) was observed with consortium. To check the effect of dye and treated samples on plant growth, the relative sensitivity of the three dyes and samples of treated wastewater with respect to Raphanus sativus was evaluated. The variation in percent germination (65-100%), length of plumule (2.71±0.19-5.28±0.31) and radicle (1.23±0.33-3.55±0.42) of R. sativus was observed in decolorized samples of different bacterial treatments. However, it was comparatively higher than that of parent dye solutions. The results suggested that the toxic nature of the degraded metabolites were less than the original dyes. xiiiAfter preliminary batch tests confirmation of superior dye degradation capacity of bacterial isolates, color removal efficiency of the bacterial consortium was evaluated a in submerged membrane bioreactor (sMBR). The laboratory-scale MBR, with hollow fiber PVDF membrane module (Water Q Ltd. Netherlands) having a working volume of approx. 2 L, was operated at 37±1 0C in a thermostatic cabinet using simulated textile wastewater under non sterile conditions for 30 days. The MBR system showed very high efficiency in terms of COD removal which was generally higher than 94% in all the reactor systems for the treatment of RB 221, RY 145, RR 195 and a mixture of the dyes. However, the COD removal efficiency of the reactor operated at different hydraulic retention times (HRT) varied. The decolorization efficiency of MBR operated at HRT-72 hrs for all three dyes was in the range of 93-99% in stable phase. In the next phase of the research, a high throughput pyrosequencing technology was adopted to investigate the community profile of the reactor during treatment of the different dyes. The significant increase in number of operational taxonomic units (OTUs) was observed upto the end of reactor operation. The results of pyrosequencing revealed overall dominance of Proteobacteria (79.125%) followed by Firmicutes and Bacteroides in all samples. The bacterial strains, which were constituents of the consortium, belonged to phylum Proteobacteria and Firmicutes. Among the consortial bacterial strains, P. aeruginosa was observed as the most dominant and was efficiently involved in the dye decolorization process, as it was recovered from different samples of reactor treating textile dyes. However, as the sterile conditions were not maintained during the reactor operation, all reactors were invaded by different groups of microorganisms. The stable color and COD removal was achieved even in the presence of invading microorganism, which indicated the involvement of invaders in dye degradation process. The bioreactor set up used for removal of reactive dyes from simulated textile effluent using developed bacterial consortia was proven to be an efficient, environmental friendly, cost effective and stable system playing an important role in removal of dye color and COD by improving biodegradability of dyes.