Study On The Role Of Extracellular Proteins During The Aerobic Sludge Granulation

Aerobic granular sludge has been demonstrated the application potential in the biological treatment of municipal and industrial wastewaters due to its excellent settleability, high pollutant removal capacity and strong resistance to toxicity. Extracellular polymeric substances(EPS), as the predominant components, play an important role in the formation and stability of aerobic granular sludge. However, the key components and mechanism of EPS on the aerobic sludge granulation is still unclear. The differences in EPS components between flocs and granules, variation of extracellular protein (PN) during the aerobic sludge granulation and toxic stress, and optimization of operational condition from the perspective of PN are investigated in this study, via the combination of spectrometric analysis, PCR-DGGE and two-dimensional electrophoresis (2-DE). Main conclusions are as follows:1、Activated sludge (flocs), aerobic granular sludge and anaerobic granular sludge (granules) were studied, and the contents and constituents of LB-EPS (loosely bound EPS, LB-EPS) and TB-EPS (tightly bound EPS, TB-EPS) were analyzed. It was found that PN contents in LB-EPS and TB-EPS of the aerobic and anaerobic granules were33.6±9.7and96.8±11.9,27.1±2.8and61.6±4.2mg-g’VSS, respectively, which were much higher than8.5±1.5and43.1±2.7of the flocs; While extracellular polysaccharide (PS) contents in these three sludges were all about30mg·g-1VSS, quite lower than PN contents. The analysis of sludge surface charge indicated that it had a linear correlation with PN content, which suggested the importance of PN in granules. The study of three-dimensional fluorescence (EEM) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) further demonstrated that PN molecular weight in flocs was mainly distributed in14.3-66.2kDa while it was20.1-97.4kDa in granules, and the proportion of PN with high molecular weight (HMW) became larger; The aromatic protein-like and tryptophan protein-like substances largely existed in granules, suggesting they were the key PN components in the structural maintenance of granules.2、Based on the aerobic granular sludge reactor, the variations of granule characteristics and PN in the process of aerobic granulation and4-chloroaniline (4-ClA) stress were studied. The results showed that during the aerobic granulation, PN content increased from69.4±8.1mg·g-1VSS to281.5±16.0mg·g-1VSS accompanied by a more obvious growth of PN with HMW in44.3-97.2kDa. Step increasing the influent4-ClA loading to0.36kg·m-3·d-1, the granules kept stable and PN increased from213.0±10.26mg·g-1VSS to390.9±60.5mg·g-1VSS; But the granular sludge disintegrated when the loading was over0.4kg·m-3·d-1, and PN decreased to171±12.6mg·g-1VSS rapidly. SDS-PAGE and Fourier transform infrared spectroscopy (FTIR) results further implied that macromolecular PN (44.3-66.4kDa), protein secondary structure amide I with3-turn helix and B-sheet, aspartate-like and tyrosine materials largely or only existed in the stable granules, suggesting they were the key components in the structural stability of granules. The analysis of microbial community structure found that the microbial abundance declined in the unstable granules under4-ClA stress and the disappearance of dominant bacteria Ornithinicoccus, Kineosphaera limosa, Diaphorobacter related to toxicity resistance, EPS production and simultaneous nitrification and denitrification might cause the breakdown of granules.3、Aiming at the stability loss of the aerobic granules in long-term running, the variations of system performance and PN during the process optimization through the united adjustment of main parameters like hydraulic retention time (HRT) and sludge settling time. The results showed after reducing HRT from17.2h to11.4h, both the sludge concentration and settleability improved, but the granulation was not obvious. In order to promote the sludge retention, the sludge settling time was prolonged from3min to10min, and the removal efficiency of4-ClA and COD reached above99%, sludge concentration kept at6.0g·L-1. FTIR and2-DE results showed that PN increased from159.7±0.1mg-g-1VSS to300±13.0mg-g-1VSS significantly during the process optimization, while the specific compositions in PN appeared to have a apparent succession and the protein secondary structure amide I with B-sheet which favored the sludge granulation increased. The study of the periodic change of pollutant removal, sludge properties and EPS contents before and after the process optimization showed that PN served as the carbon source and energy source for the microbes in the aerobic starvation phase, could improve the cell surface hydrophobicity and reduce the surface charge, thus favoring the stability of granular sludge under the optimized condition.