Title: Elucidating Microbial Community Structure, Function and Activity in Engineered Biological Nitrogen Removal Processes using Meta-omics Approaches
Description: Biological nitrogen removal (BNR) has been applied for more than a century in the interests of preserving and enhancing public health and the environment. But only during the last few decades has the development of molecular techniques using biomolecules such as nucleic acids (DNA and RNA) and proteins allowed the accurate description and characterization of the phylogenetic and functional diversity of microbial communities. Moreover, thanks to recent advances in genomics and next-generation sequencing technologies, microbial community analyses have initiated a new era of microbial ecology. Notwithstanding the fact that the efficiency and robustness of a wastewater treatment mainly depend on the composition and activity of BNR communities, research on the structural and functional microbial ecology of the engineered BNR process remains rare with respect to next-generation sequencing and bioinformatics. This dissertation aims to bridge high-priority knowledge gaps in determining and applying knowledge of microbial structure (who is there and how many?) and function (what are they doing? what else can they do?) to the practice of BNR processes, and to opening up the ‘black-box’ of energy and resource efficient engineered BNR processes using a systems biology approach.

Specific objectives were to (1) selectively enrich Nitrospira spp. from a mixed environmental microbial consortium (such as activated sludge) in a continuously operated bioreactor and characterize the microbial ecology during the course of enrichment, determine key kinetic parameters of enriched Nitrospira spp., (2) examine the inhibitory effects of nitrogenous intermediates (such as hydroxylamine, presented herein) on the physiological and molecular responses of Nitrospira spp. in terms of both catabolism and anabolism, (3) characterize bacterial community composition and their dynamics by 16S rRNA gene amplicon sequencing under varying reactor operational conditions from full-scale WWTPs and identify process parameters that most significantly correlate with those dynamics, (4) interpret metagenomic (DNA-based) and metatranscriptomic (RNA-based) derived structure, metabolic function and activity of the full-scale BNR microbial communities, and (5) describe gene expression in the same full-scale BNR communities in response to alternating anoxic-aerobic conditions using a metatranscriptomic approach.

First, planktonic Nitrospira spp. were successfully enriched from activated sludge in a sequencing batch reactor by maintaining sustained limiting extant nitrite and dissolved oxygen concentrations for a half year. The determined parameters collectively reflected not just higher affinities of this enrichment for nitrite and oxygen, respectively, but also a higher biomass yield and energy transfer efficiency relative to other NOB such as Nitrobacter spp. Used in combination, these kinetic and thermodynamic parameters can help toward the development and application of energy-efficient biological nutrient removal processes through effective Nitrospira out-selection. Second, using quantitative activity measurements (respirometrc rates) with functional gene expression profiles, this study demonstrated that N-intermediates such as hydroxylamine (NH¬2OH) can strongly inhibit the activity and expression of key anabolic (energy synthesis) and catabolic (biomass synthesis) pathways of Nitrospira spp.

A strategy that relies upon the transient accumulation and consumption of such intermediates (such as transient aeration) could provide the platform for successful suppression of Nitrospira spp. in the next generation of energy efficient engineered BNR processes. Third, 16S rRNA gene amplicon sequencing revealed that microbial community structure and their dynamics significantly varied depending on seven differing wastewater treatment processes. The findings showed that five process parameters of wastewater influenced the dynamics of BNR communities; water temperature was correlated most strongly to the variance of bacterial communities, followed by effluent NH3, effluent NO3-, removed N, and effluent NO2-. The results provided insights into the underlying ecological pattern of community compositions and dynamics in full-scale WWTPs; and correlation with process parameters brought about distinct communities that enable different microbial activities. However, one of the greatest challenges was to elucidate the relationship between microbial structure and their “active” functions, which are related to reactor performance (This challenge continued into fourth study chapter summarized below).

Fourth, continuing from the previous study, combined metagenomics and metatranscriptomics revealed far superior richness of information of not just microbial structure, but also potential (through metagenomics) and expressed function (through metatranscriptimics) within the complex activated sludge processes. Via independent analysis of whole-DNA and whole-RNA, the entire microbial community and its in situ active members, involved in nitrificaiton and denitrification, were compared. Active nitrifiers and denitrifiers obtained by RNA analysis exhibited relatively high abundances in DNA-derived communities. Further gene expression annotation on nitrogen removal revealed that the expressions of denitrification-related genes except nos were increased under anoxic conditions relative to aerobic conditions, while the expressions of nitrifying genes were decreased. Our findings led to an improved understanding of metabolic activities and roles of BNR microbial communities, and offer the first metatranscriptional insights on engineered nutrient removal in anoxic conditions relative to aerobic conditions in full-scale wastewater systems.

In sum, next-generation sequencing as well as traditional molecular techniques shed light on microbial diversity and different functional genes in varying engineered BNR systems. Furthermore, this dissertation provides a wealth of knowledge on systematic explorations of the linkage between structure and function of BNR communities, and offers engineering applications to BNR processes including energy and resource efficient engineered systems. It is expected that the implementation and further expansion of this work will improve the design and operation of engineered BNR processes, eventually producing benefits for the global population and the environment.