Abstract

Conventional biological nitrogen removal (BNR), comprised of nitrification and denitrification, is traditionally employed in wastewater treatment plants (WWTPs) to prevent eutrophication in receiving water bodies. More recently, the combination of selective ammonia to nitrite oxidation (nitritation) and autotrophic anaerobic ammonia oxidation (anammox), collectively termed deammonification, has also emerged as a possible energy- and cost-effective BNR alternative. Herein, we analyzed microbial diversity and functional potential within 13 BNR processes in the United States, Denmark, and Singapore operated with varying reactor configuration, design, and operational parameters. Using next-generation sequencing and metagenomics, gene-coding regions were aligned against a custom protein database expanded to include all published aerobic ammonia oxidizing bacteria (AOB), nitrite oxidizing bacteria (NOB), anaerobic ammonia oxidizing bacteria (AMX), and complete ammonia oxidizing bacteria (CMX). Overall contributions of these N-cycle bacteria to the total functional potential of each reactor was determined, as well as that of several organisms associated with denitrification and/or structural integrity of microbial aggregates (biofilm or granules). The potential for these engineered processes to foster a broad spectrum of microbial catabolic, anabolic, and carbon assimilation transformations was elucidated. Seeded sidestream DEMON® deammonification systems and single-stage nitritation-anammox moving bed biofilm reactors (MBBRs) and a mainstream Cleargreen reactor designed to enrich in AOB and AMX showed lower enrichment in AMX functionality than an enriched two-stage nitritation-anammox MBBR system treating mainstream wastewater. Conventional BNR systems in Singapore and the United States had distinct metagenomes, especially relating to AOB. A hydrocyclone process designed to recycle biomass granules for mainstream BNR contained almost identical structural and functional characteristics in the overflow, underflow, and inflow of mixed liquor (ALT) rather than the expected selective enrichment of specific nitrifying or AMX organisms. Inoculum used to seed a sidestream deammonification process unexpectedly contained <10% of total coding regions assigned to AMX. These results suggest the operating conditions of engineered bioprocesses shape the resident microbial structure and function far more than the bioprocess configuration itself. We also highlight the advantage of a systems- and metagenomics-based interrogation of both the microbial structure and potential function therein over targeting of individual populations or specific genes.

Highlights

  • Engineered biological nitrogen removal (BNR) processes employ mixed microbial communities for removing nitrogenous pollutants from wastewater to prevent eutrophication in receiving water bodies

  • Between 66.9 and 92.1% of filtered merged reads aligned to the custom-expanded BLASTX database, representing the total coding DNA sequences (CDS) in each metagenome, and 8.02–19.74% of filtered merged reads aligned to coding regions produced by N-transforming microorganisms selected for in-depth analysis (Table 1 and Supplementary Table 1)

  • The first category included nitritation-anammox systems, as represented by the DEMON, moving bed biofilm reactors (MBBR), and Cleargreen systems

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Summary

Introduction

Engineered biological nitrogen removal (BNR) processes employ mixed microbial communities for removing nitrogenous pollutants (ammonia, nitrite, nitrate) from wastewater to prevent eutrophication in receiving water bodies. Through suppression of NOB activity to prevent competition for nitrite as a substrate, partial nitritation (the incomplete oxidation of ammonia to nitrite) by AOB can be combined with anammox in a process referred to as deammonification to significantly reduce required oxygenation and inorganic carbon usage (Ahn et al, 2008). Complete ammonia oxidation (comammox) to nitrate in a single organism (CMX) rather than a mixed community of AOB and NOB has been recently characterized (Daims et al, 2015; van Kessel et al, 2015). The ubiquity of CMX bacteria in a variety of full-scale wastewater treatment processes has been shown using shotgun metagenomics (Annavajhala et al, 2018)

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