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

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. 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). 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. 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. 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