Ammonia-oxidizing archaea in engineered biofiltration systems
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Ammonia is a nitrogenous metabolic waste product that is produced by all animal life. High concentrations of ammonia are toxic to animals and may result in algal blooms and eutrophication in aquatic environments. To prevent negative impacts on animal and environmental health, water treatment systems use biological filters to host populations of nitrifying microorganisms that oxidize ammonia to nitrite and subsequently to nitrate. Ammonia-oxidizing archaea (AOA) outnumber ammonia-oxidizing bacteria (AOB) in many terrestrial and aquatic environments, but few studies have characterized AOA in engineered environments, despite the importance of these systems for ecosystem health. This thesis research examined two types of nitrifying biofiltration systems, including freshwater aquaria and fixed-film reactors from a municipal wastewater treatment plant (WWTP), to investigate the abundance, diversity, activity, and ecology of AOA in freshwater engineered systems. Although nitrification is the primary function of aquarium biofilters, few studies have investigated the microorganisms responsible for this process in aquaria. Based on quantitative PCR (qPCR) for ammonia monooxygenase (amoA) and 16S rRNA genes of Bacteria and Thaumarchaeota, AOA were numerically dominant in 23 of 27 freshwater biofilters, and contributed all detectable amoA genes in 12 of these biofilters. AOA also outnumbered AOB in five of eight sampled marine aquarium biofilters. For freshwater aquaria, the proportion of amoA genes from AOA relative to AOB was inversely correlated with ammonia concentration, suggesting an adaptation to low ammonia conditions. Composite clone libraries of AOA amoA genes revealed distinct freshwater and saltwater clusters, as well as mixed clusters containing both freshwater and saltwater amoA gene sequences. From one analyzed freshwater aquarium biofilter that demonstrated a high archaeal abundance, AOA representative Candidatus Nitrosotenius aquariensis was enriched in laboratory culture. Ca. N. aquariensis oxidized ammonia stoichiometrically to nitrite with a concomitant increase in thaumarchaeotal cells. Ca. N. aquariensis has a generation time of 34.9 hours, is mesophilic with an optimal growth temperature of 33ᵒC, and can tolerate up to 3 mM NH4Cl. Transmission electron microscopy (TEM) revealed that Ca. N. aquariensis cells are rod-shaped with a diameter of ~0.4 µm and lengths ranging from 0.6-3.6 µm. In addition, these cells possess paracrystalline S-layers and up to five appendages per cell. Phylogenetically, Ca. N. aquariensis belongs to the Group I.1a Thaumarchaeota, and clusters with environmental sequences from freshwater aquarium biofilters, aquaculture systems, and wastewater treatment plants (WWTPs). The complete genome sequence is 1.70 Mbp, and encodes genes involved in ammonia oxidation, urea hydrolysis, and bicarbonate assimilation. Several genes encoding flagella synthesis and chemotaxis were identified in the genome, as well as genes associated with S-layer production, defense, and protein glycosylation. Incubations of aquarium filter biomass revealed that PTIO is strongly inhibitory of ammonia oxidation, suggesting an in situ role for Ca. N. aquariensis-like AOA in freshwater aquarium biofilters. AOA have been detected in WWTPs based on targeted gene sequences, but contributions of AOA to ammonia oxidation in WWTPs remain unclear. In this thesis, ammonia-oxidizing populations in nitrifying rotating biological contactors (RBCs) from a municipal WWTP were investigated. Individual RBC stages are arranged in series, with nitrification at each stage contributing to an ammonia concentration gradient along the flowpath. Quantitative PCR for thaumarchaeotal amoA and 16S rRNA genes in RBC biofilm samples demonstrated that AOA abundance increased as ammonia decreased across the RBC flowpath. In addition, a negative correlation (R2=0.51) existed between ammonia concentration of RBC-associated water samples and the relative abundance of AOA amoA genes detected in corresponding biofilm samples. A single AOA population was detected in the RBC biofilms and this phylotype shared low 16S rRNA and amoA gene homologies with existing AOA cultures and enrichments. From RBC biofilm, Ca. Nitrosocosmicus exaquare was enriched in laboratory culture. Ca. N. exaquare oxidizes ammonia to nitrite stoichiometrically and assimilates bicarbonate, as demonstrated by microautoradiography. The 2.99 Mbp genome of Ca. N. exaquare encodes pathways for ammonia oxidation, bicarbonate fixation, and urea transport and hydrolysis. Despite assimilating inorganic carbon, the ammonia-oxidizing activity of Ca. N. exaquare is greatly stimulated in enrichment culture by the addition of organic compounds, especially malate and succinate. Ca. N. exaquare cells are coccoid and large in comparison to all other cultured AOA, with a diameter of approximately 1-2 µm. Phylogenetically, Ca. N. exaquare belongs to the Group I.1b Thaumarchaeota, clustering in the Nitrososphaera sister cluster, which includes most other environmental AOA sequences from municipal and industrial WWTPs. Incubations of WWTP biofilm demonstrated partial inhibition of ammonia-oxidizing activity by PTIO, suggesting that Ca. N. exaquare-like AOA contribute to nitrification in situ. Interestingly, CARD-FISH-microautoradiography revealed no incorporation of bicarbonate by Ca. N. exaquare-like AOA in actively nitrifying biofilms, suggesting that these cells may assimilate non-bicarbonate carbon sources. In natural and engineered environments, differential inhibitors are important for assessing the relative contributions of microbial groups to biogeochemical processes. For example, PTIO is a nitric oxide scavenger used for the specific inhibition of nitrification by AOA. This research investigated four alternative nitric oxide scavengers for their ability to differentially inhibit AOA and AOB in comparison to PTIO. Caffeic acid, curcumin, methylene blue hydrate, and trolox all demonstrated differential inhibition on laboratory cultures of AOA and AOB, providing support for the proposed role of nitric oxide as a key intermediate in the thaumarchaeotal ammonia oxidation pathway. Overall, this research demonstrated that AOA were abundant in aquarium biofilters and nitrifying RBCs, and that they contributed to ammonia-oxidizing activity in sampled biofilm environments. Niche partitioning of AOA and AOB was observed based on environmental ammonia concentrations, with AOA adapted to low ammonia conditions. Moreover, the enrichment cultures and genome sequences of novel AOA representatives provide insight into the ecophysiology of AOA originating from engineered systems.
Cite this work
Laura Sauder (2016). Ammonia-oxidizing archaea in engineered biofiltration systems. UWSpace. http://hdl.handle.net/10012/11128