Microbial Structure and Function of Engineered Biological Nitrogen Transformation Processes: Impacts of Aeration and Organic Carbon on Process Performance and Emissions of Nitrogenous Greenhouse Gas Ariane Coelho Brotto Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Graduate School of Arts and Sciences COLUMBIA UNIVERSITY 2016 © 2016 Ariane Coelho Brotto All rights reserved ABSTRACT Microbial Structure and Function of Engineered Biological Nitrogen Transformation Processes: Impacts of Aeration and Organic Carbon on Process Performance and Emissions of Nitrogenous Greenhouse Gas Ariane Coelho Brotto This doctoral research provides an advanced molecular approach for the investigation of microbial structure and function in response to operational conditions of biological nitrogen removal (BNR) processes, including those leading to direct production of a major greenhouse gas, nitrous oxide (N2O). The wastewater treatment sector is estimated to account with 3% of total anthropogenic N2O emissions [1]. Nevertheless, the contribution from wastewater treatment plants (WWTPs) is considered underestimated due to several limitations on the estimation methodology approach suggested by the Intergovernmental Panel on Climate Change (IPCC)[2]. Although for the past years efforts have been made to characterize the production of N2O from these systems, there are still several limitations on fundamental knowledge and operational applications. Those include lack of information of N2O production pathways associated with control of aeration, supplemental organic carbon sources and adaptation of the microbial community to the repeated operational conditions, among others. The components of this thesis, lab-scale investigations and full-scale monitoring of N2O production pathways and emissions in conjunction with meta-omics approach, have a combined role in addressing such limitations. Lab-scale experiments imposing short-term anoxic-aerobic cycling on partial- and full- nitrification based processes were conducted to investigate the microbial response to N2O production. Interestingly, it was determined that full-nitrification systems could be a higher contributor to N2O production and emissions than partial-nitrification. While it has been reported in the literature a higher contribution from the latter when the microbial community is not subjected to oxygen cycling conditions [3]. Following the knowledge obtained with a single anoxic-aerobic cycle imposed to nitrifying communities, long-term adaptation of the microbial community to continued anoxic-aerobic cycling and its impact on N2O production were investigated through a meta-omics approach. Long-term studies are particularly significant regarding engineered systems, where the microorganisms are continually subjected to cycling conditions again and again. A microbial adaptation at the RNA level was identified on both autotroph and heterotroph organisms. The transcripts of the metabolic pathways related to NO and N2O production (nir, nor) and consumption (nor, nos) were initially induced followed by a gradual decline, leading to a parallel reduction in gaseous emissions over time. Other pathways not typically interrogated in conjunction with the nitrogen metabolism, such as electron transport chain and carbon fixation were also investigated and revealed a mechanism to overcome the imbalance in electron flow and generation of proton motive force (increased transcription of terminal oxidase genes, cco and cox) to uphold carbon fixation during continued cycling. The second part of this thesis focuses on full-scale WWTPs, where it is crucial to determine specific nuances of the systems’ dynamics and of the different types of treatment that may contribute to increased production and emissions of N2O. For that purpose, two distinct BNR systems not usually considered and studied in terms of N2O production and emissions were chosen. First, a separate centrate treatment (SCT) process employing glycerol as the supplemental carbon source was monitored. Significantly, this system was found to have one of the highest levels of N2O production and emission report thus far [4-6]. Glycerol revealed to foster a microbial community (i.e. Burkholderiales, Rhodobacterales and Sphingomonadales) that stores internal carbon and promote partial denitrification, leading to accumulation of nitrite and N2O [7-11]. Second, both fixed- and moving-bed biofilm BNR systems were investigated. The overall N2O emission fractions for the Integrated Fixed-Film Activated Sludge (IFAS)(0.09 – 1.1% infl-TKN) and denitrification filters (0.11 – 1.4% infl-TN) were similar to the reported emissions from suspended growth activated sludge systems [4-6]. For the IFAS system, aqueous and gaseous N2O profiles paralleled the diurnal variability on influent nitrogen load. The production of N2O was significantly correlated with ammonia concentration (p<0.05, r=0.91), suggesting the production through hydroxylamine oxidation pathway. Denitrification filters displayed a very peculiar pattern on N2O emissions associated with intermittent operational cycles (i.e. nitrogen release cycle and backwash). These intrinsic operations of the denitrification filters contributed to transient oxygen conditions and nearly the entire N2O emissions through gaseous stripping and production by inhibition of denitrification. Similarly to suspended growth systems, process design and operations demonstrated to also play an important role in N2O emissions from attached growth processes. Finally, aeration strategies for energy efficient conventional nitrification based on the microbial community development and its associated performance was investigated in lab-scale. It was demonstrated that using the same air supply rate, continuous and intermittent aeration resulted in completely different microbial structure. Consequently, distinct kinetics and nitrification performance were observed. The aeration rate could be minimiZed (resulting in reduction in energy consumption) for high ammonia removal efficiency and lower N2O emissions, as long as the process is designed accordingly to the microbial ecology developed in such conditions. In sum, the microbial structure, function and connection of metabolic pathways of complex engineered microbial communities as applicable to BNR systems and its operations were investigated in detail. From an engineering perspective, this dissertation provides an advancement on the molecular approach to characterize structure and function of microbial responses to engineered operations beyond the business-as-usual target genes, which can eventually result in better design and control of engineered BNR processes. This study offers more than an improved scientific understanding of the complex microbial environment and direct engineering applications. It connects sanitation with water quality and the greenhouse gas effect by prioritiZing concurrent enhanced biological nitrogen removal and mitigation of N2O production and emission. Ultimately the implications of the result presented herein can provide economical, environmental, health benefits for the society. TABLE OF CONTENTS LIST OF FIGURES ..................................................................................................................... vi LIST OF TABLES ....................................................................................................................... ix LIST OF ABBREVIATIONS AND SYMBOLS ........................................................................ x CHAPTER 1 INTRODUCTION .................................................. ............................................... 1 1.1. Engineered Nitrogen cycle ................................................................................................ 1 1.2. Relevance of N2O to the environment .............................................................................. 3 1.3. Microorganisms involved in N2O production.................................................................. 5 1.3.1.Ammonia oxidiZing bacteria .......................................................................................... 5 1.3.2. Ordinary heterotrophic microorganisms ....................................................................... 8 1.3.3. Other microorganisms ................................................................................................... 9 1.4. Biological nitrogen removal systems .............................................................................. 11 1.5. Nitrous oxide production pathways in BNR wastewater treatment systems ............. 13 1.5.1. Implication of microbial activity and electron flow balance on N2O production ....... 14 1.5.2. Low dissolved oxygen concentration.......................................................................... 17 1.5.3. Transient conditions .................................................................................................... 18 1.5.4. Nitrite accumulation.................................................................................................... 20 1.5.5. Effect of carbon sources (COD/N ratio) during chemoorganoheterotrophic denitrification ........................................................................................................................ 21 1.5.6. Effect of inorganic carbon limitation on ammonia oxidiZing bacteria ....................... 22 1.5.7. Physical process .........................................................................................................