Studies On Biodegradation Of Phenolic Hyper-saline Wastewater And Dynamics Of Microbial Community Structure During The Treatment Process

The source of phenolic wastewater is extensive and the compositions are complicated, such as coking,pharmaceutical,chemical and dying wastewatwer.The existence of high concentration of phenolics and salts in wastewater will inhibit the growth of the microorganism and brings more difficulty in biological treatment,which has become a difficult problem in domestic and international wastewater treatment field and is urgent to be solved currently.To solve the problem of inhibitory effect of high salt on traditional biological treatment system,the objectives of this paper are to explore of salt-tolerant biological treatment systems,which are also capable of degrading phenolics,study on the characteristics of treatment systems under different conditions and analyze the community structure during treatment process by molecular biology technology,such as RISA(Ribosomal intergenic sequence analysis),AFDRA(Amplified functional DNA restriction analysis) and 16S rDNA sequencing analysis.The experimental results will clarify the relationship between the microstructure and macrofunction of treatment system and provide a theoretical and technical supports for the biological treatment of phenolic hyper-saline wastewater.The activated sludge could degrade different phenolic mixtures under high salt conditions. Inhibitory effect of catchol and salicylate on phenol degradation was not obvious,but the removal rate was decreased in the presence of p-cresol.Salinity and pH fluctuation would affact the removal effeciency of activated sludge.When the content of NaCl was lower than 50 g/L, the removal rate of phenolics was increased with the NaCl concentration increasing,but decreased in the presence of more than 100 g/L NaCl.The efficiency of degradation was stable under neutral conditions(pH 7.0-9.0).At the same time,the microbial community structure of activated sludge was analyzed by RISA and 16S rDNA sequencing to clarify the relationship between the microstructure and macrofunction of treatment system.The RISA patterns revealed that the diversity of microorganism was increased in single substrate system,but was little change in binary substrates system.The community structure was drastically change during salt fluctuation process,but remained stable during pH fluctuation.16S rDNA sequence analysis showed that Halomonas sp.and Marinobactergenus sp.were predominant species during salt fluctuation process,which have better ability of salt tolerant.Marinobacter sp.and Alcaligenes faecalis sp.were the main species during the pH and substrate concentration fluctuation process.It was also found that Shewanella sp.and Arthrobacter sp.were existence in treatment system.The changes of phenol hydroxylase gone of activated sludge was analyzed by AFDRA under different conditions,which revealed the change of microbial community structure and function of activated sludge in molecular level.AFDRA on phenol hydroxylase genes show that the diversity of phenol hydroxylase genes was substantially different in various conditions, such as different substrate,concentration,salinity and pH,and the changes are consistent to that of microbial community.Thus,it was concluded that the changes of functional genes could reflect the dynamics of microbial community,and the microbial community structure could affect the diversity of functional genes.Arthrobacter sp.W1,Rodococcus sp.W2 and Candida sp.W3 was isolated from the activated sludge and could utilize phenol as sole carbon source under high salt conditions, which was identified on the basis of physiological and biochemical tests and 16S rDNA and 26S rDNA sequence analysis.The sequence of strains were submitted to GenBank with the number EU339930,EU339931 and EU349016.The degradation and growth conditions of three strains were as follows:temperature 20-30℃,pH 5.0-9.0 and salinity 10-100 g/L NaCl. They could utilize benzoic,salicylate,p-cresol,aminophenol and hydroquinone as the carbone source.Under high salt conditions,strains were mainly accumulated compatible solutes(ectoine and betaine) in cells to resist the extracellular hyper-saline environment. Adding compatible solutes such as ectoine,betaine and glutamic acid could improve the growth of strains under high salt conditions and enhance the salt-tolerance ability.A defined microbial consortium was constructed through orthogonal experiments by using of strain W1,W2 and W3.The adamptation conditons for the phenol degradation by the consortium were as followed:temperature 10-40℃,pH 4.0-12.0,salinity 10-150 g/L NaCl. Compared to other three pure strains,the consortium could degrade phenol under severe conditions and had a wider substrates range.During degradation of mixed phenolics by the consortium in SBR,the treatment efficiency was stable under different conditions in 3 monthes.Compared with the activated sludge system,the consortium showed higher removal efficiency and resistant ability of environmental fluctuation more powerful.Fluorescence in situ hybridization(FISH) analysis indicates that the abundance and intesnsity of specific probe of Arthrobacter sp.W1 was more than other two strains when salinity and substrate concentration fluctuation.During pH fluctuation process,there was lilltle differences of abundance and intensity of specific probe among three strains.It means that Arthrobacter sp.W1 has resistant ability of salinity and substrate concentration fluctuation more powerful,and three strains are insensitive to pH fluctuation.The results of real-time quantitative PCR showed that the cell numbers of Arthrobacter sp.W1 was increased fromel.16×10¹³ to 5.71×10¹³ cell per g of dry weight when NaCl concentration raised from 10 to 100 g/L.The cell number was change between 1.47×10¹³ to 5.53×10¹³ cell per g of dry weight when substrated concentration fluctuation process,but the cell numbers always remained stable during pH fluctuation process.Thus,it was concluded that W1 was the most important microorganism of the consortium during degradation phenolic hyper-saline wastewater process,which maintained higher metabolic activity under fluctuate conditions.