Different Efficiency Mechanism Of Co-metabolic Decolorization And Degradation Of Azo Dye By The Functional Flora/Strains With Sugar Sources

Azo dyes are the most widely used synthetic dyes.However,they are reported to be carcinogenic and mutagenic.It is therefore imperative to develop affordable and effective technologies to enable a sufficient decolorization and degradation of dyestuffs and their intermediates remaining after the processing of textiles in printing and dyeing wastewater.Up to now,extensive researches have dealt with the important role of bacteria in biological treatment processes of dyes.The application of a bacterial consortium has advantages because of its strong adaptability and complementing catabolic diversity of different microorganisms.Co-metabolism,as a promising biotechnology,has been applied to boost the biological performance for the removal of refractory pollutants.The addition of optimal co-substrates,especially sugar sources,to the microbial systems has been reported to enhance strongly the biological treatment of azo dyes.However,only few researches reported about comparative studies of these interrelated sugar sources as co-substrates and even less about the different efficiency mechanism and the intrinsic molecular mechanism.Consequently,three kinds of carbohydrates related to each other at the metabolic level were selected to combine into four different sugar sources as co-substrates in this paper,namely: sucrose(disaccharide,which was capable of decomposing into one molecule of glucose and one molecule of fructose),glucose(monosaccharide),fructose(monosaccharide),glucose+fructose.A series of static batch experiments were carried out under microaerophilic(static)conditions,in which the natural bacterial flora DDMZ1 and the high-toxicity dis-azo dye reactive black 5(RB5)were chosen as the flora source and the target pollutant,respectively.RB5 degradation kinetics,azoreductase activities,microbial community structures,the differential expression mechanism of functional genes and proteins were comprehensively studied using sugar sources for co-metabolism.The main research works in this study are listed as follows:(1)Screening of functional flora and study on its biological characteristicsThe functional flora DDMZ1 capable of decolorizing and degrading RB5 was a natural mixed bacterial flora domesticated from the activated sludge by concentration gradient training method.In this group rod-shaped bacteria were the dominant ones.The effects of culture methods,p H,temperatures,salt concentrations and RB5 initial concentrations on the decolorization ability of bacterial flora DDMZ1 were investigated to analyze its biological characteristics.The results indicated that the flora DDMZ1 achieved the highest decolorization and degradation effiency on RB5 at the optimal conditions(p H=5.5,37 °C).Additionally,the flora DDMZ1 showed the salt tolerance to some extent,and still exhibited high decolorization and degradation ability to high concentrations of RB5.(2)Different efficiency mechanism of decolorization and degradation of RB5 by the functional flora with different sugar sourcesHere we investigated the different efficiency mechanism of using four interrelated sugar sources(glucose,fructose,sucrose,glucose+fructose)as co-substrates for the decolorization and degradation of RB5 by the flora DDMZ1.The effects of four sugar sources on the degradation kinetics,azoreductase activities,degradation products and microbial community structures were comprehensively studied.The results indicated that four interrelated sugar sources were served as promising co-substrates to promote the decolorization and degradation of RB5 by the flora DDMZ1.Among these,fructose was the most effective one.In four sugar sources co-metabolism systems,the removal of RB5 with different initial concentrations followed the first-order kinetic model.Four sugar sources stimulated the azoreductase activities,and the sample with fructose exhibited the highest azoreductase activity.LC-TOF-MS results indicated that the dye RB5 was biodegraded into compounds with smaller molecular weight and simpler structure in the presence of four sugar sources.Fructose addition leaded to the formation of more metabolites in comparison with the other three sugar sources samples.High-throughput sequencing results showed that fructose caused the enrichment of genus Klebsiella in addition to the apparently dominant genus Burkholderia-Paraburkholderia.It was speculated that these two dominant genera might play important roles in the decolorization and degradation of RB5 by the flora DDMZ1.(3)The genome and proteome response mechanism of the functional flora using fructose for co-metabolismThe genome and proteome response mechanism and the intrinsic boosting mechanism of decolorization and degradation of RB5 by the flora DDMZ1 using fructose(the optimal sugar source)for co-metabolism were systematically studied.A metagenomic sequencing approach was applied to perform the species annotations and functional annotations of genes,and then the genome response mechanism of the flora DDMZ1 was analyzed.The genes encoding NADH: ubiquinone reductase,NADPH: quinone reductase and FMN-dependent NADH-azoreductase were considered as to play a crucial role in biodegradation process of RB5.A label-free quantitative proteomic approach was used to verify the up-regulated key proteins/enzymes encoded by functional genes.According to GO annotations,some of the up-regulated proteins were related to reductase activities,ring cleavage and intermediate transmembrane processes for RB5 biodegradation.The pathway for benzoate degradation was enriched and considered to be the most connected pathway according to the KEGG analysis.Several up-regulated oxidoreductases,dehydrogenases/reductases and membrane proteins might play essential roles during the biodegradation of RB5 in the fructose co-metabolism system.The combinatorial profiling of metagenomics and metaproteomics indicated that the GO and KEGG annotations presented great correlations between gene and protein levels.Based on the correlation of the expression of functional genes and functional proteins in the dominant genera Pseudomonas and Burkholderia,the overall molecular degradation mechanism for RB5 was proposed.(4)Isolation,identification of functional strains and study on their biological characteristicsTwo functional strains named Burkholderia strain DDMZ1-1 and Pseudomonas aeruginosa strain DDMZ1-2 were isolated from the functional flora DDMZ1.The effects of p H,temperatures and salt concentrations on the decolorization ability of two functional strains were investigated to analyze their biological characteristics.The results indicated that two functional strains achieved the highest decolorization and degradation efficiency on RB5 at the optimal conditions(p H=5.5,37 °C).However,they were both lower than the decolorizing effect of flora DDMZ1.In addition,two functional strains showed the salt tolerance to some extent,which could be applied to the treatment of dyeing wastewater with high salinity.The addition of fructose could significantly enhance the decolorization performance and azoreductase activities of two functional strains with respect to different initial concentrations of RB5,which was consistent with the previous effect of fructose on the flora DDMZ1.A quantitative analysis of the selected azoreductase gene azo R3 by real-time PCR showed that the quantitative value of azo R3 in Pseudomonas aeruginosa strain DDMZ1-2 was much higher than that in Burkholderia strain DDMZ1-1.The addition of fructose could significantly stimulate the expression of this azoreductase gene.Additionally,fructose as co-substrate could strongly enhance the decolorizing effect of RB5 by two functional strains with different concentration ratios.Two functional strains also exhibited strong broad-spectrum degradation performance on different structural dyes.This paper revealed the functional flora/strains,key enzyme activities,functional genes,functional proteins and their interactions with sugar sources,which were linked mutually at the metabolic level and caused the differences of co-metabolic efficiency.The study provided new ideas for bioremediation of refractory organic pollutants,and at the same time provided a theoretical basis for in-situ bioaugmentation and effective regulation of azo-dye-containing wastewater biological decolorization processing.