Oligotyping and Genome-Resolved Metagenomics Reveal DistinctCandidatusAccumulibacter Communities in Full-Scale Side-Stream versus Conventional Enhanced Biological Phosphorus Removal (EBPR) Configurations

Abstract

21Candidatus Accumulibacter phosphatis (CAP) and its sub-clades-level diversity has been 22 associated and implicated in successful phosphorus removal performance in enhanced biological 23 phosphorus removal (EBPR). Development of high-throughput untargeted methods to 24 characterize clades of CAP in EBPR communities can enable a better understanding of 25 Accumulibacter ecology at a higher-resolution beyond OTU-level in wastewater resource 26 recovery facilities (WRRFs). In this study, for the first time, using integrated 16S rRNA gene 27 sequencing, oligotyping and genome-resolved metagenomics, we were able to reveal clade-level 28 differences in Accumulibacter communities and associate the differences with two different full-29 scale EBPR configurations. The results led to the identification and characterization of a distinct 30 and dominant Accumulibacter oligotype -Oligotype 2 (belonging to Clade IIC) and its matching 31 MAG (RC14) associated with side-stream EBPR configuration. We are also able to extract 32 MAGs belonging to CAP clades IIB (RCAB4-2) and II (RC18) which did not have 33 representative genomes before. This study demonstrates and validates the use of a high-34 throughput approach of oligotyping analysis of 16S rRNA gene sequences to elucidate CAP 35 clade-level diversity. We also show the existence of a previously uncharacterized diversity of 36 CAP clades in full-scale EBPR communities through extraction of MAGs, for the first time from 37 full-scale facilities. 38 Introduction 39Enhanced biological phosphorus removal (EBPR) has been a promising technology for 40 phosphorus removal from municipal wastewater, due to its sustainable nature and P recovery 41 potential in comparison to chemical precipitation and adsorption-based approaches [1]. However, 42 conventional EBPR processes face challenges related to system stability and susceptibility to 43 variations and fluctuations in influent loading conditions such as unfavorable carbon to P (C/P) 44 ratios [2]. A new emerging process, namely, side-stream EBPR (S2EBPR) features an anaerobic 45 side-stream reactor that allows a portion of the return activated sludge (RAS) to undergo 46 hydrolysis and fermentation thus enabling influent carbon-independent enrichment and selection 47 of polyphosphate accumulation organisms (PAO). Facilities that have operated or piloted 48 S2EBPR showed improved and more stable performance [3][4][5][6]. However, several key 49 knowledge gaps still exist for this process including lack of understanding of the fundamental 50 mechanisms governing the process and differences in microbial ecology, particularly 51 functionally relevant key populations such as PAOs between the conventional and the S2EBPR 52 processes. Several studies that have characterized the microbial ecology of different process 53 configurations using 16S rRNA gene amplicon sequencing, fluorescence in-situ hybridization 54 (FISH) and quantitative polymerase chain reaction (qPCR) have failed to associate differences in 55 PAO microbial ecology with proc...

Combined Enhanced Biological Phosphorus Removal (EBPR) and Nitrite Accumulation for Treating High-strength Wastewater

“…By phylogenetic comparison with the previous study from our group and the literature, the results revealed that the dominant oligotype 1 in our reactor was highly consistent with the specific Accumulibacter oligotype 2 (belonging to Clade ⅡC) accounting for 62.4-68.0% in the full-scale side-stream EBPR system (highlighted in Fig. 6B), which was observed to be much lower (<20%) in the conventional EBPR-A2O system (Srinivasan et al, 2019).…”

“…(A) Relative abundance of oligotypes of 16S rRNA gene amplicon sequences classified as Candidatus Accumulibacter at different stages of the lab-scale reactor operation; (B) Phylogenetic tree of Candidatus Accumulibacter oligotype representative sequences in our system (in red), oligotypes curated in the previous study from full-scale side-stream EBPR sludge (Srinivasan et al, 2019) (in blue) and reference sequences of Candidatus Accumulibacter phosphatis from the MiDAS database (McIlroy et al, 2015) (in black).…”

“…Oligotyping was carried out to further resolve Candidatus Accmulibacter genus OTUs into sub-genotypes, namely oligotypes, based on the high-variational sites in reconstructed 16S sequences, following the protocol in previous studies (Eren et al, 2013, Srinivasan et al, 2019). To reveal the phylogeny of identified Accumulibacter oligotypes, a phylogenetic analysis was conducted on all identified oligotype representative sequences of our reactor (3 total), Accumulibacter oligotypes in a previous study from our group (9 total) (Srinivasan et al, 2019) and Accumulibacter phosphatis reference sequences in the MiDAS database (18 total) (McIlroy et al, 2015). An extra random sequence ( Dechloromonas , FLASV96.1460) in the same Rhodocyclaceae family was chosen from the MiDAS database as outgroup.…”

Modeling Versatile and Dynamic Anaerobic Metabolism for PAOs/GAOs Competition Using Agent-based Model and Verification via Single Cell Raman Micro-spectroscopy

“…S2EBPR refers to modified EBPR configurations that include diversion of a portion of RAS or anaerobic mixed liquor to a side-stream reactor, where simultaneous VFA production via sludge hydrolysis and fermentation and PAO activity-related P release and carbon uptake occur. Compared to conventional EBPR design, the S2EBPR offers a suite of advantages including influent-carbon independent condition for PAO enrichment that eliminates the influences of fluctuating influent loads, more controllable lower-redox environment with more complex VFA composition that provides more favorable selection of PAOs over GAOs, flexible implementation configurations and potential reduction of carbon footprint and denitrification enhancement by diverting more influent carbon to denitrification 1,9,11,[19][20][21][22] ..…”

Prospects for multi-omics in the microbial ecology of water engineering

“…Genomes of multiple Ca. Accumulibacter strains have since been assembled to date Camejo et al, 2019;Flowers et al, 2013;Gao et al, 2019;Mao et al, 2014;Qiu et al, 2020;Skennerton et al, 2015), including high-quality genomes assembled from full-scale WWTPs (Law et al, 2016;Srinivasan et al, 2019), even though it remains to be isolated in pure-culture. The uncultivated bacterium Kuenenia stuttgartiensis (Strous et al, 2006) underlying anaerobic ammonium oxidation (anammox) was assembled from a complex bioreactor community, and subsequently other anammox species within the Planctomycetes have been assembled (Lawson et al, 2017;Speth et al, 2016).…”