Oligotyping and metagenomics reveal distinct Candidatus Accumulibacter communities in side-stream versus conventional full-scale enhanced biological phosphorus removal (EBPR) systems

Highlights

• Distinct Accumulibacter communities were identified in two different EBPR processes.

• Oligotyping can reveal microdiversity in Accumulibacter communities.

• Three new Accumulibacter MAGs were recovered from full-scale EBPR processes.

• Two of the MAGs belong to clades without any previous representative genomes.

Abstract

Candidatus Accumulibacter phosphatis (CAP) and its clade-level micro-diversity has been associated with and implicated in functional differences in phosphorus removal performance in enhanced biological phosphorus removal (EBPR) systems. Side-stream EBPR (S2EBPR) is an emerging process that has been shown to present a suite of advantages over the conventional EBPR design, however, large knowledge gaps remain in terms of its underlying ecological mechanisms. Here, we compared and revealed the higher-resolution differences in microbial ecology of CAP between a full-scale side-stream EBPR configuration and a conventional A2O EBPR process that were operated in parallel and with the same influent feed. Even though the relative abundance of CAP, revealed by 16S rRNA gene amplicon sequencing, was similar in both treatment trains, a clade-level analysis, using combined 16S rRNA-gene based amplicon sequencing and oligotyping analysis and metagenomics analysis, revealed the distinct CAP microdiversity between the S2EBPR and A2O configurations that likely attributed to the improved performance in S2EBPR in comparison to conventional EBPR. Furthermore, genome-resolved metagenomics enabled extraction of three metagenome-assembled genomes (MAGs) belonging to CAP clades IIB (RCAB4-2), IIC (RC14) and II (RC18), from full-scale EBPR sludge for the first time, including a distinct Ca. Accumulibacter clade that is dominant and associated only with the S2EBPR configuration. The results also revealed the temporally increasing predominance of RC14, which belonged to Clade IIC, during the implementation of the S2EBPR configuration. Finally, we also show the existence of previously uncharacterized diversity of clades of CAP, namely the clades IIB and as yet unidentified clade of type II, in full-scale EBPR communities, highlighting the unknown diversity of CAP communities in full-scale EBPR systems.

Introduction

Enhanced biological phosphorus removal (EBPR) has been a promising technology for phosphorus removal from municipal wastewater, due to its sustainable nature and P recovery potential in comparison to chemical precipitation and adsorption-based approaches (Morse et al., 1998). However, conventional EBPR processes face challenges related to system stability and susceptibility to variations and fluctuations in influent loading conditions such as unfavorable carbon to P (C/P) ratios (Gu et al., 2008). A new emerging process, namely, side-stream EBPR (S2EBPR) features an anaerobic side-stream reactor that allows a portion of the return activated sludge (RAS) to undergo hydrolysis and fermentation thus enabling influent carbon-independent enrichment and selection of polyphosphate accumulating organisms (PAO). Facilities that have operated or piloted S2EBPR showed improved and more stable performance (Barnard and Kobylinski, 2018; Gu et al., 2019; Onnis-Hayden et al., 2018; Wang et al., 2019). However, our understanding of the fundamental mechanisms governing the process and differences in microbial ecology, particularly functionally relevant key populations such as PAOs between the conventional and the S2EBPR processes remain unclear. This hinders its wider applications to realize its full benefits. The activated sludge process (particularly biological nutrient removal) is intricately linked to microbial community dynamics through microbial kinetics and stoichiometry. The link between the microbial ecology and process performance (kinetics and stoichiometry) is fundamental to improved process design and operation. Several studies that have characterized the microbial ecology of different process configurations, using 16S rRNA gene amplicon sequencing, fluorescence in-situ hybridization (FISH) and quantitative polymerase chain reaction (qPCR), were not able to reveal major differences in PAO microbial ecology between S2EBPR and conventional configurations (Mielczarek et al., 2013; Stokholm-Bjerregaard et al., 2017). This is, at least partially due to the complex and overwhelming “masking” effect of full-scale facility-specific selection forces (i.e. varying influent characteristics, site-specific environmental and operational conditions etc.) exerted on the microbial community structure, and the potential lack of sufficient resolution in methods of analysis which makes it very difficult to isolate the effect of process configuration alone.

Candidatus (Ca.) Accumulibacter phosphatis (hereafter referred to as Accumulibacter or CAP) is considered to be a key PAO in both lab-scale and full-scale EBPR systems and its finer-resolution-diversity seem to have implications in their EBPR-relevant functions (Albertsen et al., 2012; Barr et al., 2016; Flowers et al., 2013; Martín et al., 2006; Oyserman et al., 2016b; Skennerton et al., 2015). Our current understanding of the physiology, metabolic potential, transcriptional, and proteomic dynamics are based on lab-scale Accumulibacter-enriched reactors from which 19 metagenome-assembled genomes (MAGs) of Accumulibacter have been obtained (Albertsen et al., 2016; Camejo et al., 2019a; Mao et al., 2014; Martín et al., 2006; Parks et al., 2017; Skennerton et al., 2015). Amongst them, only one complete draft genome sequence for Clade IIA, namely Accumulibacter UW1 has been reported. These MAGs have highlighted the enormous genomic diversity in Accumulibacter clades (>700 unique genes in each clade) especially in terms of the capacity to perform the different steps of the denitrification pathway and ability to metabolize complex carbon sources (Skennerton et al., 2015). Metagenomic studies have indicated high-level of diversity within Accumulibacter populations in full-scale EBPR communities (Albertsen et al., 2012; Law et al., 2016), however this diversity was not fully characterized due to their low relative abundance and the inability to extract high-quality MAGs. This phylogenetic diversity within CAP populations along with their previously indicated possible functional diversity across types and clades point towards ecological niche-partitioning due to determinants such as type of carbon source, phosphorus levels, length of anaerobic period, and presence of alternate electron acceptors such as nitrate and nitrite etc. High-throughput characterization of functionally relevant populations at the appropriate resolution can provide a better understanding of microbial dynamics and metabolic processes which, in turn, can lead to better process engineering.

Current methods to characterize clade-level differences in Accumulibacter populations include qPCR, FISH, ppk1 gene sequencing and metagenomic sequencing. qPCR and FISH have been extensively used to characterize the clade-level differences in lab-scale and full-scale systems (Camejo et al., 2016; He et al., 2007; Mao et al., 2015; Welles et al., 2017). However, they target only those currently known clades for which primers or probes are available. Metagenomic and recovery of ppk1 gene sequences from metagenomic data are capable of providing phylogenetic information, however they are cost-prohibitive and/or time-consuming for routine analysis at full-scale practice. Although, 16S rRNA gene amplicon sequencing has emerged to be a high-throughput and widely-used method to characterize and capture broad shifts in microbial communities, it is recognized that it often fails to capture the phylogenetic diversity within OTUs (also called microdiversity) (Chase, 2018; Edgar, 2018). Recent work has argued for the use of a higher similarity threshold (Chase, 2018; Edgar, 2018) or for the use of exact sequence variants (ESVs) or amplicon sequence variants (ASVs) or oligotypes (Callahan et al., 2017; Eren et al., 2013) to more accurately characterize the microdiversity in a community. Oligotyping, a method to obtain ASVs, has been proposed as a method that can distinguish meaningful sequence variation from sequencing errors and can partition sequence types that differ in as little as one nucleotide with the assumption that the variation in small regions of the 16S rRNA gene translate to phylogenetic differences (Eren et al., 2013; Fisher et al., 2015, 2014). Oligotyping has been used previously to explore the microdiversity of dominant taxa in sewage, animal fecal and fresh water lake microbiomes (Berry et al., 2017; Fisher et al., 2015, 2014). It should be noted that even though long read sequencing approaches using PacBio and Oxford Nanopore sequencing technologies are being developed and could be useful in characterizing microdiversity, their application in wastewater has not been fully evaluated and its application is still limited by error rates and limited availability of capacity.

In this study, we employed 16S rRNA gene sequencing, oligotyping and metagenomic analysis to elucidate and compare the microbial ecology and potential functional traits of Accumulibacter between conventional and S2EBPR process configurations in full-scale systems. The unique opportunity to operate two different EBPR configurations, in parallel and separate treatment trains and secondary clarifiers with the same influent feed (Gu et al., 2019; Wang et al., 2019), made it possible to eliminate the effect of influent characteristics-related selection factors on the microbial ecology and, therefore enabled evaluation and association of specific CAP populations with S2EBPR versus conventional A2O configuration. In-situ monitoring and ex-situ activity tests were used to assess the differences in performance and metabolic activity of EBPR-related populations in the process (Gu et al., 2019; Wang et al., 2019). In order to overcome the limitation of 16S rRNA gene sequencing and OTU-based approaches, we, for the first time, employed oligotyping analysis and ppk1 gene-based evaluation using ppk1 gene sequences retrieved from metagenomic sequencing to reveal the microdiversity within Accumulibacter populations. In addition, using differential coverage binning, we successfully assembled and binned 2 high-quality and 1 medium-quality Accumulibacter MAGs from full-scale EBPR sludge, including a distinct Ca. Accumulibacter clade that is dominant and associated only with the S2EBPR configuration. Lastly, comparative genomic analysis was performed with the extracted MAGs and with previously published CAP MAGs, to determine potential differences in key metabolic pathways and functions including unique functions that could contribute to niche-partitioning in the dynamic and complex environments in full-scale EBPR facilities.