Microbial ecology of nitrification in engineered water treatment systems

Abstract

Nitrification is performed primarily by chemolithoautotrophic microorganisms and is important for nitrogen transformation in aquatic environments. The understanding of nitrification has evolved over the years from a previously understood two-step process mediated by ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) that perform ammonia oxidation to nitrite, and nitrite-oxidizing bacteria (NOB) that perform nitrite oxidation to nitrate. More recent research has revealed the existence of complete ammonia-oxidizing (“comammox” or CMX) bacteria from the genus Nitrospira that are capable of oxidizing ammonia to nitrate. With three groups of ammonia oxidizers often existing in the same environment, research is needed to understand the microbial ecology of nitrifying communities. These ammonia oxidizers play important roles in engineered systems, including wastewater treatment plants (WWTP) and aquarium biofilters, where they transform ammonia waste (NH₃/NH₄⁺) to less toxic nitrate (NO₃⁻) via nitrite (NO₂⁻). Prior to the discovery of comammox Nitrospira, previous research revealed that AOA dominated over AOB in freshwater aquarium biofilters. In Chapter 2, aquarium biofilter microbial communities were profiled and the abundance of all three known ammonia oxidizers were quantified using 16S rRNA gene sequencing and quantitative PCR (qPCR), respectively. Biofilter and water samples were each collected from representative residential and commercial freshwater and saltwater aquariums. Distinct biofilter microbial communities were associated with freshwater and saltwater biofilters. Comammox Nitrospira amoA genes were detected in all 38 freshwater biofilter samples and dominant in 30, whereas AOA were present in 35 freshwater biofilter samples and only dominant in 7 of them. The AOB were at relatively low abundance within biofilters, except for the aquarium with the highest ammonia concentration. For saltwater biofilters, AOA or AOB were differentially abundant, with no comammox Nitrospira detected. Additional sequencing of Nitrospira amoA genes revealed differential distributions, suggesting niche adaptation based on water chemistry (e.g., ammonia, carbonate hardness, and alkalinity). Network analysis of freshwater microbial communities demonstrated positive correlations between nitrifiers and heterotrophs, suggesting metabolic and ecological interactions within biofilters. These results indicate that comammox Nitrospira play a previously overlooked but important role in home aquarium biofilter nitrification. Following the identification of comammox Nitrospira among dominant ammonia oxidizers in freshwater aquarium biofilters, Chapter 3 monitored microbial community succession and water chemistry for three independent home aquariums during their first 12-weeks after start-up, with weekly collection of biofilter beads and sponge samples. Extracted DNA from biofilter samples was used for 16S rRNA gene sequencing to determine microbial community composition and quantitative PCR (qPCR) to quantify ammonia monooxygenase (amoA) genes. Water samples were also collected weekly for measurements of ammonia, nitrite, and nitrate. Biofilter nitrification activity reduced ammonia and nitrite concentrations below detectable limits by week 3 in two of the three aquariums, which showed comparable nitrification activity by the week 8 time point. Detection of ammonia oxidizers by qPCR coincided with ammonia oxidation activity for all systems. The two aquariums with nitrification occurring by week 3 contained live plants, whereas the aquarium with delayed nitrification did not, suggesting that live plants might provide an effective nitrifier inoculation source for aquarium establishment. Additionally, a preference in biofilter material was observed for detected AOA, which were present in higher abundance in bead samples compared to sponge samples. Although the tested aquaria differed in the timing and prevalence of ammonia oxidizers in biofilters during community establishment, samples from all three aquaria were consistently dominated by comammox Nitrospira by the end of 12 weeks. Additional metagenomic functional profiling of week 12 biofilter samples confirmed the presence of AOA amoA genes and comammox Nitrospira amoA genes as detected by qPCR for all aquaria, with nitrite oxidation marker gene nxrB for both comammox Nitrospira spp. and canonical NOB Nitrospira spp. also detected. Although this work sheds light on how ammonia oxidizers establish in residential freshwater aquaria, further research is needed to test factors that impact the establishment of nitrifiers populations, such as inoculation sources, fish loads, and water chemistry. Novel WWTP technologies, including membrane aerated biofilm reactors (MABR), aim to improve nitrogen removal, reduce energy consumption, and improve nitrification in cold weather conditions. The municipal WWTP in Southern Ontario was upgraded with an MABR system in 2022, which was installed upstream of the existing conventional activated sludge (CAS) system at a municipal scale. This current study evaluates the impact of an MABR upgrade on the CAS and MABR microbial communities, with this MABR system being the largest in North America based on media surface area at the time of its installation. In Chapter 4, the microbial community of the CAS system was characterized before and after the MABR upgrade to evaluate the impact of the upstream MABR installation on the functional potential of the downstream activated sludge. Microbial community characterization was done using 16S rRNA gene amplicon sequencing of the V4-V5 region and additional MABR biofilm samples were characterized using metagenomic analysis to evaluate the functional potential of the biofilm for nitrification and denitrification. Before upgrade, the CAS system included the nitrifiers Nitrosomonas (AOB) and Nitrobacter (NOB), which exhibited seasonal abundance and activity differences. Following the upgrade to the hybrid MABR-CAS system, seeding effects from the MABR biofilm increased diversity of the CAS, including nitrifiers. Along with AOB, Nitrospira NOB and comammox Nitrospira were present in the MABR. Metagenomic profiling showed that biofilm microbial communities were well equipped to perform nitrification, denitrification, and phosphate removal. Characterization of the microbial communities in the plant showed that the MABR technology had a positive impact in increasing microbial diversity in this treatment system, along with an increased inventory of nitrifiers with diverse metabolic capabilities.