Compared to conventional nitrification and denitrification, anaerobic ammonium oxidation (anammox) is a more energy saving and cost effective process for biological nitrogen removal (BNR). To date, the anammox process has been applied widely and designed mainly to treat sidestream wastewaters. However, only 15%-20% of the influent domestic sewage nitrogen loading is present in the sidestream, while the bulk of it still needs to be removed from the mainstream. Research efforts thus have shifted from sidestream to mainstream applications of anammox, including the application of anammox bioreactors at low temperature, low influent ammonium strength, and under the presence of organic carbon (characteristic of municipal mainstream wastewaters). In this dissertation research, the applicability of anammox process in lab-scale and full-scale mainstream systems have been studied. The overall goals of this dissertation research were (1) to develop an effective strategy to enrich an anammox moving bed biofilm reactor (MBBR) under low influent nitrogenous substrate concentration and ambient temperature (23 Cº), and link microbial ecology to the process performance of the enriched anammox MBBR; (2) to explore the catabolism and anabolism of anammox bacteria in a mainstream MBBR before and after dosing of organic carbon; (3) to extend the strategy of mainstream anammox enrichment under ambient temperature (23 Cº) to low temperature (15 Cº) , and link microbial ecology to the process performance; (4) to evaluate the microbial community structure, kinetics and performance during startup and long-term operation of a full-scale mainstream anammox process; (5) to investigate the reliability of the new enriched mainstream anammox MBBR under the imposition of additional wet weather flow; (6) to develop a reliable and sensitive mothed of hydrazine determination in anammox reactor. First, an anammox MBBR was successfully enriched under low nitrogenous substrate and ambient temperature. It needs to be addressed that, even with the limited fraction of Candidatus “Kuenenia stuttgartiensis” in the coming inoculum from the sidestream MBBR, Candidatus “Kuenenia stuttgartiensis” was effectively enriched in the biofilm biomass of the mainstream MBBR. Moreover, the enhanced activity of Candidatus “Kuenenia stuttgartiensis” was demonstrated through this whole time series experiments, and achieved the most competitive level among all functional groups. Therefore, the importance and necessity of bioaugmentation are addressed during the enrichment of mainstream anammox process. Second, successful enrichment of a mainstream anammox moving bed biofilm reactor was accomplished at low nitrogenous substrate and low temperature. 16S amplicon sequencing was employed to investigate the microbial ecology of the biomass in the biofilm and suspension. Results showed the dominance of Candidatus "Kuenenia" related anammox bacteria in the biofilm of mainstream reactor, though Nitrospira spp. related nitrite oxidizing bacteria were still detected in a limited fraction. These results are crucial to show the effective enrichment of anammox reactor by bioaugmentation even under low temperature, especially in a practical way. Third, the performance, kinetics and microbial ecology were studied before, during and after the imposition of additional organic carbon. The dosing of organic carbon resulted in a reversible negative impact on both the activity of AMX and the reactor performance. Stable isotope probe and 16S amplicon sequencing were applied to investigate the metabolism of functional groups. The results showed anammox bacteria are not capable of assimilating acetate, while the community assimilating 13C-labeled acetate was mainly assigned to denitrifiers. Presence of denitrifiers were observed in the mainstream MBBR and stayed inactive without sufficient organic carbon. In sum, these results demonstrate that the mainstream anammox process as tested was resilient to a short period imposition of organic carbon. Fourth, the performance and microbial ecology of the ambient-temperature mainstream anammox were investigated under wet weather condition. Based on the full recovery of reactor performance as well as the stable microbial ecology, the applicability of the mainstream MBBR under wet weather conditions was demonstrated. Fifth, real-time polymerase chain reaction was applied to evaluate the startup and operation of two parallel sidestream DEMONTM systems as well as the initiation of the mainstream anammox process through bioaugmentation. Results provided the evidence that anammox bacteria was the most abundant functional group in two parallel DEMONTM systems, showing the successful startup in the sidestream. Furthermore, anammox bacteria were selectively retained in the mainstream with high bioaugmentation rates from the sidestream. These results are critical to demonstrate the significance of bioaugmentation in the startup of mainstream anammox system even in full-scale wastewater water treatment plant. Finally, a sensitive and reliable spectrophotometric method was proposed to measure hydrazine concentration in anammox reactor. The concentration of hydrazine could be precisely determined in the presence of nitrite, when a certain amount of sulfamic acid is introduced. In sum, the application and characterization of anaerobic ammonium oxidation (anammox) process to treat sidestream and mainstream wastewaters in both lab-scale and full-scale was investigated in detail. From a practical perspective, the knowledge gained can lead to a better design and operation of engineered nitrogen removal process.

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.

This study involved the use of soil and suspended growth microcosms to study the variation in groundwater denitrification rates using different substrates. Two gaseous substrates, carbon monoxide and methane, were studied and compared to a common liquid substrate, methanol. Denitrification with carbon monoxide as a substrate was achieved using an acclimated seed of mixed activated sludge and anaerobic digester sludge. Kinetic studies of denitrification using carbon monoxide suggested a strong substrate inhibition effect. The observed maximum denitrification velocity of 0.026 mg N/d-mg VSS occurred at a carbon monoxide partial pressure of 0.10 atmospheres (2.8 mg/ℓ). At higher carbon monoxide partial pressures, denitrification velocities decreased. The denitrification velocities at various carbon monoxide concentrations were described by a modified form of the Haldane substrate inhibition model. The biomass yield using carbon monoxide was 1.1 mg VSS/mg VSS, the maximum specific growth rate was 0.03 mg VSS/d-mg VSS, and the half velocity constant was 26 mg-N/ℓ. Denitrification rates using carbon monoxide as a substrate were much slower than those obtained using methanol, and the cost of carbon monoxide was much higher. Denitrification occurred readily, when methanol was provided as a substrate, in microcosms containing either a clay soil, a sandy soil, or activated sludge. Under the conditions of this study, denitrification was not achieved in clay soil or sandy soil microcosms using methane or carbon monoxide as substrates. Denitrification was not achieved using methane as a substrate with an activated sludge seed. / Ph. D.

Ph. D.