Exploring The Origin And Structural Role Of Extracellular DNA In Aerobic Granular Sludge

Aerobic granular sludge(AGS)is regarded as a promising biotechnology that can replace conventional activated sludge for both domestic and industrial wastewater treatment.Extracellular polymeric substances(EPS)consisted of polysaccharides,proteins and extracellular DNA(exDNA)are the primary factors responsible for maintaining the stability of AGS.However,current studies have focused on the aggregation of exopolysaccharides and proteins,and on studies have explored the microbial origin and structural role of extracellular DNA(exDNA)in AGS.In this study a high efficiency method to separate intracellular DNA(inDNA)and exDNA was used to assess the characteristics of exDNA in different sizes AGSs combining metagenomics analysis.ExDNA was found to have a significantly lower GC content,?46.0%,than the?65.0%GC of inDNA.Taxonomic predictions showed that the majority of the reads from the exDNA that could be taxonomically assigned were from members of the phyla Bacteroidetes(55.0?64.2%).InDNA reads that were assigned were mainly from Proteobacteria(50.9?57.8%)or Actinobacteria(18.0?28.0%).The draft Bacteroidetes genomes assembled and then binned by inDNA reads and exDNA reads are highly similar and complete,which is consistent with cell lysis as a source of exDNA.In addition,functional annotation of contigs of the Bacteroidetes genomes revealed that enrichment of CRISPR arrays in Bacteroidetes genomes associated draft genomes and CRISPR-CAS proteins in the draft genomes assemblrd and binned by exDNA reads suggested that bacteriophage infection may be an important cause of DNA release by lysis of these cells.A critical role for this exDNA was found using DNase I digestion experiments,which showed that the exDNA was vital to maintain the structural stability of relatively small sized AGS but not significant for the larger sized AGS,in addition,extended DLVO theoretical analysis revealed that exDNA can affect the aggregation behavior of microorganisms in AGS mainly by changing Lewis acid-base interaction.Specific fluorescence dyeing technique and confocal laser scanning microscopy(CLSM)were used to observe exDNA in situ.It was found that most exDNA was bound to extracellular proteins and expolysaccharides to support the structure of AGS.This study revealed the characteristics of exDNA in AGS and the role of structural support,providing a new perspective for the study on the structural stability of AGS and theoretical support for the popularization and application of AGS technology.