Metagenomic and Predictive Functional Analysis of Microbial Communities in a Novel Wastewater Treatment System

Abstract

Aerobic granulation is an emerging microbial process in wastewater treatment that has shown to improve the efficiency of conventional activated sludge systems by accelerating sedimentation, improving organic waste, nitrogen, and phosphorus removal, and increasing microbial tolerance to toxic elements found in wastewater. Aerobic granulation results in large microbial aggregates that sediment faster contribute to higher cell tolerance than bacterial flocs found in conventional AS systems. To date, granulation can only be achieved in sequence batch reactors, which are largely incompatible with the continuous flow model used in modern wastewater treatment plants. Recent research conducted at Virginia Polytechnic Institute and State University has demonstrated for the first time that a proper ratio of feast to famine conditions is able to promote aerobic granulation in a simulated plug flow reactor, composed of a suite of completely stirred tank reactors in series. Feast and famine cycles are known to negatively select for filamentous microbes that contribute to poor aggregate density in continuous flow wastewater reactors and positively select for microbes known to contribute to biofilm formation. The goal of the present study is to understand the mechanisms of aerobic granulation present in this novel reactor system by determining changes in the microbial community composition in aerobic granules compared to conventional microbial flocs. A metagenomic analysis was conducted using 16S rDNA sequencing on aerobic granules and conventional AS samples fed by the same wastewater. Taxonomic identification and predictive functional metagenomics were completed with bioinformatics software tools Bioconductor, PICRUSt2, and BURRITO. In contrast to previous metagenomic reports of AS in contemporary wastewater treatment facilities, the results of this thesis have revealed the microbial community changes and predicted functionality of bacteria in the new reactor system that efficiently facilitated the aerobic granulation process. These findings include increased prevalence of bacterial taxa such as Comamonadaceae, Hydrogenophaga, Flavobacterium and Sphingopyxis in the PFR system with aerobic granular sludge, which are known to produce extracellular polymeric substances and are commonly identified in SBRs. Additionally, drastic decreases were observed for taxa including Actinobacteria, Chloroflexi, Accumulibacter, Microthrix, and Zoogloea, which are filamentous groups largely associated with sludge bulking issues and poor sedimentation in traditional AS. Apparent variability in abundance between the chambers of the PFRs with failed granulation and successful granulation indicate higher compositional stability in AGS. Predictive functional analysis further indicates upregulation of proteins involved in biofilm formation pathways in AGS, such quorum sensing, secretion systems, and transporters. Upregulation of nitrogen and sulfur metabolism in AGS also agree with the expected activity of granular sludge communities compared to flocs in AS. Somewhat unexpectedly, growth rates and other metabolic functions are upregulated in AS, which may be explained by the stationary phenotype of a mature AGS system. These results indicate the selection of bacteria that can contribute to biofilm formation and inhibition of filamentous microbes, suggesting that the feast/famine profile in the PFR systems provided similarly adequate conditions to that of other successful SBR systems.