Ecotoxicological and Biodegradation Studies of Reactive Azo Textile Dyes using Different Microorganisms

Abstract

Reactive azo dyes are one of the most extensively used dyeing agents in the textile industry. Representatives of this class of dyes containing sulfonic acid derivatives, are usually more persistent in the aquatic environments due to their high water-solubility and complex polyaromatic nature. Due to the xenobiotic, possible toxic, and recalcitrant nature, these dyes act as one of the major environmental hazards that can adversely affect the aquatic ecology. The assessment of their toxicity in different living systems is therefore vital to document their possible role in the ecological milieus. Biological dye waste treatment strategies, employing pure or mixed microbial cultures in suspended and/or attached forms, is usually deemed as an effective, economically sustainable, and eco-efficient approach. Moreover, for the complex heterogenic natured textile effluents, application of microbes with a more adaptive physiological profile (such as the extremophilic bacteria) could provide a more workable solution. In the present sets of studies, ecotoxicity assessment and biotreatment investigations for three model reactive azo dyes i.e., Reactive Blue 221 (RB 221), Reactive Red 195 (RR 195), and Reactive Yellow 145 (RY 145) (pure and/or mixed form) were carried out using specialized microorganisms from different environmental origins in lab scale attached and suspended reactors. Treatment and detoxification efficiencies were monitored through analytical techniques and toxicity assays. Organismic-level acute toxicology profile of three reactive azo dyes and their mixture was investigated, by using bacterial (Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Listeria monocytogenes, and Bacillus subtilis), fungal (Trichoderma asperellum, Aspergillus flavus, Fusarium fujikuroi, and Rhizoctonia solani), plant (Raphanus sativus, Triticum aestivum, Sorghum bicolor, and Phaseolus mungo), and aquatic (Artemia salina and Daphnia magna) specimens. Microbial test organisms (all the six bacteria and two fungi, i.e., T. asperellum and A. flavus) and D. magna were found to be relatively more sensitive towards the reactive azo dyes and their mixture, as the EC50 values were in the range of 80-330, 135-360, and 108-242 ppm for bacteria, fungi, and D. magna, respectively (generally the effect was not acutely toxic). Moreover, the dye mixture had a comparable effect to the individual dyes in almost all the tested microbial specimens. For plant seeds, the dye mixture was found to be relatively more inhibitory towards T. aestivum and R. sativus than the individual dyes. For S. bicolor and P. mungo seeds, the effect of the dye mixture was almost identical to the individual dyes. However, in all cases, EC50 values were in the range of 950-3500 ppm, which indicates a non-toxic effect on plant seed germination potential. Likewise, the dyes and their mixture were not acutely toxic for the aquatic test specimens. Efficacy of an immobilized fungal (Trichoderma asperellum SI14) and a hybrid activated sludge reactor was evaluated and compared in batch mode for the treatment of RB 221 containing synthetic textile wastewater. Scotch Brite™ was used as support material in both setups; and the reactors’ efficacy was tested at 25, 50, and 100 ppm of RB 221 over a period of 144 hours. Both types of reactors showed 90-98% color removal with a relatively faster reduction observed with the immobilized fungal reactor (50-90% reduction in 48 hours of reactor operation in all the three batches). However, the hybrid activated sludge reactor was found to be more efficient in chemical oxygen demand (COD) reduction (79-92%) and demonstrated a smooth decline as compared to the fungal reactor, where 73-83% reduction was observed with spells of highs and lows during the treatment. Moreover, the hybrid activated sludge system showed a stable performance in terms of pH maintenance as compared to the immobilized fungal reactor, where drastic pH alterations were observed. Furthermore, a trend towards increased attached active bacterial biomass on the support material was observed through the successive batches. Biodegradation of the dye was confirmed through FTIR band changes. Both reactors did not produce toxic dye metabolites, as indicated by the phytotoxicity and brine shrimp acute toxicity analyses. Three enriched natural bacterial consortia (obtained from different locations) with different temperature, pH, and salt tolerance profiles [mesophilic [MBC], thermophilic [TBC], and alkaliphilic-halotolerant [AHBC]), were investigated for the treatment of a reactive azo dye mixture containing synthetic textile effluent. Based on the results of single factor optimization studies, the decolorization potential of the consortia was further optimized using Response Surface Methodology through Box-Behnken design (BBD). The models’ adequacy was confirmed through ANOVA, determination constant values and validation experiments. The consortia efficiently removed the dye mixture (100 ppm; glucose [sucrose for TBC], and yeast extract, 0.1% [w/v] each as co-substrates) in 72 (MBC and AHBC) and 120 hours (TBC) from the synthetic effluent under the optimal conditions. For MBC and AHBC, maximum activity was observed around mesophilic temperature conditions i.e., 35.51 and 34.93 ℃, respectively; while 50.81 ℃ was found as the suitable temperature for TBC. The optimal pH for both types of consortia was neutral. However, with AHBC the dye mixture effluent was decolorized, under high pH (10.08) and salinity (10.68% [w/v]) conditions. Additionally, up to 70% COD reduction was observed with all the three consortia in ~144 hours. Through 454-pyrosequencing, a complex community of anaerobic, facultative, and aerobic bacteria with some efficient dye degraders were found in all the three consortia. Bacteria with different temperature tolerance abilities (mesophilic and thermophilic) were isolated for the treatment of reactive azo dyes. Through the biochemical and molecular characterization (16S rRNA gene analysis) the mesophilic isolates were identified as: Pseudomonas aeruginosa NHS1, Pseudomonas sp. NHS2, Escherichia sp. NHS3, and Escherichia coli NHS4. The thermophilic isolates were: Aeribacillus pallidus, Aeribacillus sp., Geobacillus sp. NHT3, and Brevibacillus borstelensis NHT4. Cocultures were designed and optimized through OFAT and RSM using BBD design layout. The mesophilic and thermophilic cocultures decolorized the 100 and 50 ppm dye mixture in the presence of co-substrates (0.1% [w/v) yeast extract and glucose, each) in 24 and 48 hours, respectively. The mesophilic coculture showed optimal performance at temperature: 34.75 ℃, pH: 7.69, and dye concentration: 111 ppm. Whereas, 50.25 ℃, 7.31, and 60 ppm, were the most suitable temperature, pH, and dye concentration values for the dye removal by the thermophilic coculture. Through first order kinetics, growth and degradation rates for the individual and mixed dyes were computed and modeled, which showed one phasic growth and degradation process except for the dye mixture removal by the mesophilic pure cultures and coculture, where a two-phasic dye removal system was involved. Furthermore, mesophilic bacteria were relatively more efficient and showed a higher dye tolerance than the thermophiles. Biodegradation analysis of the treatment systems involving natural and designed consortia was done through FTIR spectral and GC-MS analyses. With the natural consortia, the dye mixture was converted to low molecular weight long chained aliphatic and aromatic compounds. However, the cocultures (designed consortia) converted the individual dyes and their mixture into low molecular weight aliphatic and aromatic products. The metabolites produced from both the natural and designed consortia were not toxic towards plant seeds and brine shrimp larvae, which confirms the eco-safety of these treatment methods.