Sammendrag
The effluent from industries and urbanization contains phosphorus, which pollutes water bodies and depletes phosphorus sources. To address this problem, the Hias method is considered an advanced and effective technique for phosphorus recovery and removal, combining Enhanced Biological Phosphorus Removal (EBPR) and Moving Bed Biofilm Reactor (MBBR) systems. This study aims to determine the diversity of polyphosphate-accumulating organisms (PAO) Candidatus Accumulibacter in the Hias biofilms using phosphate kinase (ppk1) gene as a genetic marker. Eight primer sets targeting the ppk1 gene were designed for the PCR amplification of samples taken from Hias biofilm, with the optimum temperature determined using gradient PCR. Pfam and phylogenetic analysis were performed to examine the conserved domains and evolutionary relationships of eight Ca. Accumulibacter sequences obtained through metagenomic studies. TOPO-TA cloning and sanger sequencing were conducted for the identification and validation amplified products. Finally, RT-qPCR was performed to determine the ppk1 expression of Accumulibacter species involved in EBPR using the expression level of the ppk1 gene across different zones of the bioreactor. The results indicate that all analyzed Ca. Accumulibacter sequences contain similar domain architecture of polyphosphate kinase domains, with their phylogeny revealing the existence of four distinct clade with high confidence. Sanger sequencing of ppk1 gene fragments from two cloning reactions resulted in high-quality sequences, showing over 85% identity with the Ca. Accumulibacter Phosphatis sequence validating the presence of species abundantly in zone 4. RT-qPCR analysis revealed fluctuating Ct values across reactor zones, indicating upregulation of the ppk1 gene in anaerobic and downregulation in aerobic zone. This study contributes to the effective phosphorus removal by analysing the complex diversity of PAO Ca. Accumulibacter Phosphatis and phosphorus removal efficiency by ppk1 gene. It is concluded that, Ca. Accumulibacter Phosphatis was the largely abundant P accumulation bacterial group in Hias process. This explains the intricate diversity of Hias samples and necessitates further analysis of ppk1 transcript dynamic across other PAO strains and biofilm samples.
