Study On The Role Of Signal Molecules AHLs In Aerobic Sludge Granulation

Aerobic granular sludge(AGS)technology has a wide application prospect in wastewater treatment because of its high treatment efficiency,low operating cost and small occupation.The formation and stabilization of AGS are regulated by the signal molecules of quorum sensing system.It is crucial to study the role of signal molecules on the formation and stabilization of AGS from the perspective of microbiology.In order to explore the role of signal molecules N-Acyl homoserine lactones(AHLs)in the formation of AGS,this study established the pretreatment and detection method of AHLs based on UHPLC-QTOF-MS,studied the changes of AHLs during the formation of AGS,and analyzed the inherent relationship between AHLs and sludge characteristics,extracellular polymeric substances(EPS)secretion,and microbial community.The results are as follows:(1)Through the optimization of chromatographic conditions,extraction methods,eluent and operation conditions,a method for simultaneous determination of 11 AHLs in phenolic wastewater was established.It showed that the solid phase extraction with methanol as eluent can achieve optimum pretreatment effect,and the recovery and accuracy of AHLs can be improved by slowly passing through 200 m L sample under gravity,eluting with 10 m L methanol,and constanting the volume to 2 m L.Under the optimized conditions,the linearity of standard curves for 11 AHLs was great,with R~2greater than 0.99.The limits of detection and quantification were 20～500 ng/L and500～2000 ng/L,respectively.The recoveries ranged from 82.21%to 146.53%,and the relative standard deviation(RSD)were less than 7.88%.This method has great accuracy and precision,which was less affected by complex matrix and suitable for real sample analysis.(2)AGS was successfully formed in a sequencing batch reactor(SBR)for the treatment of phenolic wastewater.The reactor operated stably within 35 days,and the removal rates of chemical oxygen demand(COD)and phenol were over 95%and 98%,respectively.The average granular size of AGS was up to 0.373 mm.During granulation,the relative hydrophobicity of sludge was around 80%,the EPS content increased from 45.65 mg/g VSS to 149.39 mg/g VSS,and the ratio of extracellular protein to extracellular polysaccharide(PN/PS)increased from 0.07 to 1.24.The total AHLs in both sludge and water phase reached largest value on 14th day,which were280.74 ng/g VSS and 378.63 ng/L,respectively.During the formation of AGS,the microbial community structure changed significantly.Luteococcus,Thiothrix and Norrank＿f＿sapospiraceae were the dominant genera in granules.(3)During sludge granulation,the relative content of PS and PN increased in flocculent sludge and small granules,respectively.The proportion of AHLs in granules was higher,and most of which were exsited in small granules.The relative abundance of AHLs producing bacteria Rhodobacter was highest in small granules.It was indicated that AHLs mainly played roles in the early and middle stages of granular formation,regulating the secretion of PN and promoting the growth of small granules into large one.The relative abundance of EPS secreting bacteria belonging to Zoogloea,UKL13-1,Comamonas and Flavobacterium was high in both flocculent and small granules,indicating that microorganisms with EPS-secreting function were enriched to promote the formation of AGS.The microbial diversity and richness in large granules were highest,and in which also enriched Pseudomonas,Thauera and other genera with denitrification function.(4)By analyzing the correlation between AHLs and sludge properties,EPS and microbial community,it was found that C4-HSL,3-oxo-C6-HSL,3-oxo-C10-HSL and3-oxo-C14-HSL were the key AHLs to regulate EPS secretion during the formation of AGS.AHLs in sludge phase were closely correlated with EPS-secreting bacteria,while AHLs in water phase were mainly correlated with denitrifying bacteria.Slow-growing microorganisms such as Candidatus＿Competibacter and Micropruina were closely correlated with the granular size and sedimentability of AGS.Through functional gene prediction and analysis,it was found that AGS had higher abundance of functional genes of metabolism,genetic information processing and environmental information processing.Based on the above analysis,the mechanism of AHLs on the formation and stabilization of AGS was proposed.