The Mechanism Of Microbial Quorum Sensing During The Process Of Aerobic Sludge Granulation And Stabilization

Aerobic granular sludge?AGS?is a promising technology for biological wastewater treatment,and has the advantages of compact structure,excellent settleability,high biomass and abundant microbial community.This technology has the potential to improve the sludge liquid separation,enhance the pollutants removal performance and decrease the project investment.Thus,AGS is expected to overcome the existing issues of biological wastewater treatment,i.e.,unstable effluent quality,higher energy consumption and large footprint.Nevertheless,unstable structure and limited operation have been the main bottleneck for the industrial application of AGS.Considering that the microbial aggregation and steady metabolism are the basic for the formation and stability of AGS,it is necessary to investigate the mechanism of aerobic sludge granulation and structure stabilization in the aspect of microbial aggregation,extracellular polymeric substances?EPS?secretion,substrate transformation and biochemical energy production.The aim of this thesis is to enhance the formation and stable structure of AGS.The regulation of quorum sensing on EPS secretion,substrate transformation and metabolisms and succession of microbial community is carried out.The main results are as follows:1. The physical characteristics,key EPS components and microbial community succession of AGS were investigated under different sludge retention time?SRT?.The results illustrated that the granular size was more than 500?m and the TN removal efficiency reached 85%when the SRT was maintained at 6 days,which were greater than the results of control group.When the SRT was maintained at 6 days,the extracellular protein?PN?content gradually increased from 151.6±10.5 to 247.3±12.7mg/g VSS with the sludge granulation,and the key PN components were tryptophan and protein-like substances.At the same time,the exopolysaccharides?PS?content increased from 38.4-48.3 mg/g VSS during the initial period of aerobic sludge granulation to 96.8±6.7 mg/g VSS with granular maturation,and the key PS components were alginate-like exopolysaccharides?ALE?.The results of microbial community showed that Rhodobacteraceae?28.6%?,Xanthomonadaceae?18.6%?,and Hyphomonadaceae?3.9%?were the dominant microbes in stable granules under short SRT,higher than those in the other two reactors significantly?p<0.05?.The AHLs in AGS system was analyzed,and results showed that the concentrations of N-octanoyl-DL-homoserine lactone?C8-HSL?,N-?3-hydroxydodecanoyl?-L-homoserine lactone?3OHC8-HSL?and N-?3-hydroxydodecanoyl?-DL-homoserine lactone?3OHC12-HSL?increased with aerobic sludge granulation,and were maintained at 6285.9±320.7,1325.0±148.5 and256.6±15.5 ng/L,these were the main AHLs in AGS system.Among them,the concentrations of AHLs with 8-carbon sidechain,like C8-HSL and 3OHC8-HSL,were positive correlated with the content of tryptophan and protein-like substances significantly?p<0.01?.The results initially revealed that the richness of Rhodobacteraceae and Xanthomonadaceae favored the regulation of EPS secretion and the formation of AGS.2. The novel operation mode of aeration reflux was built based on enhanced EPS secretion in view of the binding between EPS and Ca.The results showed that under the novel operation mode,MLSS of sludge was 6800 mg/L,the AGS were compact?Integrity coefficient<0.1?,and Ca²⁺content in AGS was maintained at 45-57 mg/g VSS,higher than control group of 27-42 mg/g VSS significantly?p<0.05?.Moreover,The proportion of GG block in ALE was greater than 60%,higher than control group of45.3±0.1%.The results of microbial community further showed that Rhodobacter spp.,unclassified Rhodobacteraceae and unclassified Xanthomonadaceae with the function of AHLs production were enriched in granules cultivated under aeration reflux mode,with relative abuandance of 2.5%,17.1%and 11.7%respectively.At the same time,the concentration of AHLs mentioned in previous section?C8-HSL,3OHC8-HSL and 3 OHC12-HSL?increased.The Ca-ALE combination would be achieved efficiently in granules via aeration reflux mode,which favored effective colonization of functional microbes,enhanced EPS secretion and long-term stability of AGS3. The regulation of quorum sensing for EPS yield and substrate transformation were studied.The results showed that the EPS content in AGS cultivated with complex substrate?soluble starch?as the main carbon greater than that cultivated with sodium acetate as the main carbon source.Moreover,the PN content in the granules cultivated with soluble starch increased from 93.9±1.6 mg/g VSS in initial period to 172.0±1.2mg/g VSS in stable period,and the PS content was stable at 84.6-97.9 mg/?g?VSS?.Principal component analysis?PCA?revealed that C8-HSL,3OHC8-HSL and 3 OHC12-HSL were still the main factor for the stability of aerobic granular sludge cultivated with different carbon sources.At the same time,these AHLs were positive correlation with MLSS and EPS component significantly?p<0.05?.The AHLs added-back study illustrated that the substrate degradation rate and EPS increment rate increased with C8-HSL and 3OHC8-HSL added.Especially soluble starch as the main influent carbon source,the substrate degradation rate were 0.024-0.029 mg COD/?L?min?higher than that of control group of 65%-85%Moreover,increment rate of PN and PS was 30%-36%and 25%-29%.The results revealed that the AHLs-based quorum sensing enhanced the stability of aerobic granular sludge via facilitating hydrolysis of substrate and EPS secretion?mainly PN?.4. The enhancements of quorum sensing for the microbial energy generation,EPS secretion and AGS stability were investigated.The stable and disintegrated AGS were analyzed and results showed that the concentrations of the main AHLs?C8-HSL,3OHC8-HSL,3OHC12-HSL?,ATP and EPS content decreased when AGS disintegrated.And the AHLs-producing microbes,Xanthomonadaceae?unclassified and Rhodobacter spp.were initially washed out from the reactor.The AHLs added-back study revealed that the ATP content reached 2.6±0.2?mol/g VSS with AHLs added,higher than that of control group?ATP content of 0.2±0.01?mol/g VSS?.At the same time,the addition of AHLs facilitated the increment of EPS content significantly?p<0.05?,especially tryptophan and protein-like with the relative fluorescence intensity of 745.3?Control group was only 456.3?.When the ATP synthesis was limited,EPS secretion was limited obviously with AHLs addition.The maximum content of PN and PS was 39.2±1.2 and 22.5±4.3 mg/g VSS respectively,lower than the control group without limitation of ATP synthesis?The corresponding PN and PS content was 41.2±1.9 and 25.3±0.3 mg/g VSS?.It was concluded that the AHLs-based quorum sensing favored EPS secretion via the enhancement of ATP synthesis,and the stability of aerobic granular sludge was achieved.In summary,quorum sensing enhanced the EPS secretion effectively via regulating substrates transformation and microbial metabolism,and the enrichment of microbes and AGS stability were achieved.This study revealed the mechanisms of quorum sensing for granular formation and its stability in the aspect of microbial metabolism,and provided the novel viewpoint and idea for the full-scale application of AGS.