Ecology of nitrifiers within engineered freshwater systems

Abstract

Home aquarium systems and wastewater treatment plants (WWTPs) are engineered freshwater systems that rely on nitrifying microorganisms to remove ammonia and nitrite, which are harmful to aquatic life. In aquaria, inadequate nitrification can lead to ammonia/nitrite accumulation and fish mortality. Similarly, WWTPs must ensure removal of these nitrogen species prior to effluent discharge to prevent eutrophication of downstream streams and lakes. Despite their importance, nitrifying microbial succession in aquarium biofilters is poorly documented in peer-reviewed literature, and the diversity of WWTP designs and operations complicates broad generalizations about nitrifier activity within these engineered systems. Understanding how nitrifiers establish and persist within aquarium biofilters can provide insight into nitrifier ecology but may also be useful for developing effective home aquarium nitrifying supplements and guiding best practices for aquarium ammonia management. Likewise, examining nitrifier activity in engineered wastewater systems, including tertiary treatment processes such as rotating biological contactors (RBCs), can guide strategies to enhance nitrogen removal and optimize treatment performance. Newly established home aquarium systems have an increased risk of ammonia and nitrite accumulation, and associated fish toxicity, because they lack an established nitrifying guild. This problem can be further exacerbated by increased fish loads, because more fish will produce more metabolic waste and ammonia. Thus, the first goal of this thesis was to investigate how fish load influences microbial community succession and associated nitrifying populations. Analyzing aquarium biofilter media and water samples obtained previously, ammonia and nitrite concentrations were significantly higher in tanks with higher fish loads, although biofilters associated with all treatments depleted ammonia within similar timeframes. Filter microbial community profiles were influenced by fish load, with aquaria under higher fish loads showing increased relative abundances of biofilm-associated taxa, including Planctomycetes members, whereas aquaria under low fish loads contained more abundant Gammaproteobacteria populations. After six months, aquarium microbial communities differentiated based on fish load, with low fish-load aquarium microbial community composition resembling the early- to mid-stage timepoints of the high fish-load aquaria. Nitrifying populations were similar overall, with high relative abundances of Nitrospira spp. dominated by comammox Nitrospira. Aquaria with increased fish loads contained more abundant populations of ammonia-oxidizing bacteria (AOB) compared to aquaria with fewer fish, likely perhaps resulting from the increased ammonia load. Ammonia-oxidizing archaea (AOA) were undetected in all filter microbial community profiles following data processing, which likely may reflects reflect a lack of inoculation given their commonplace abundance in most home aquarium biofilters. Together, these results formalize how fish loads affect ammonia accumulation in home aquarium systems and how nitrifier establishment and succession is linked to the number of fish housed within freshwater aquarium systems. Many home aquarists minimize ammonia and nitrite accumulation in newly established aquaria by using nitrifying supplements, which help to augment filter-associated nitrifiers. These supplements contain nitrifying populations, but research on their microbial community composition and in situ effectiveness is lacking. The second goal of this thesis was to test enrichment cultures of comammox Nitrospira (CX) and the AOA Ca. Nitrosotenuis aquarius (AQ) for use as aquarium nitrifying supplements and to evaluate their efficacy alongside an existing conventional nitrifying supplement (CS). Experiments involving new aquaria investigated how supplements altered early and post-establishment ammonia accumulation and oxidation rates. The results demonstrate that maximizing supplement dosage was important for ensuring effective oxidation of ammonia to nitrate. Treating aquaria with CX, either as the sole supplement or in combination with AQ or CS, mitigated ammonia and nitrite accumulation and maintained ammonia and nitrite at low concentrations when aquaria were treated with the highest dose. Whereas Nitrosospira sequences associated with CS were rarely detected within biofilter profiles, those associated with CX and AQ established early and persisted temporally. Treating aquaria with CX also provided extended protection against nitrite accumulation during acute ammonia-loading events. Overall, these results highlight potential benefits of using CX as a home aquarium nitrifying supplement and demonstrate how nitrifying guilds might be differentially adapted for home aquarium conditions. The Guelph WWTP contains a tertiary treatment system composed of RBCs that was previously shown to host abundant Nitrospira populations, and less abundant but temporally persistent populations of AOA Ca. Nitrosocosmicus hydrocola. The third goal of this thesis was to investigate the microbial activity transcriptional activity of this system, focusing on nitrifier-associated transcriptional expression to identify nitrifying contribution and potential novel metabolisms. Metatranscriptomic and 16S rRNA gene surveys of the Guelph WWTP RBCs reinforced dominance of abundant and active Nitrospira populations. Transcripts associated with ammonia and nitrite oxidation were among the most abundantly expressed for Nitrospira, in addition to moderate expression of putative ureases, urea transporters, and agmatinases/guanidinases/arginases, suggesting that Nitrospira may generate ammonia from multiple nitrogenous compounds in ammonia-limited environments. Some of the most abundantly expressed transcripts affiliated with Ca. N. hydrocola were associated with a putative guanidinase, suggesting that AOA may be capable of degrading nitrogen rich compounds, such as guanidine, agmatine, or arginine, to release urea and ammonia. These results lay the foundation for future research to confirm whether Ca. N. hydrocola is able to supplement nitrification with guanidine-derived ammonia. Investigating nitrifiers within engineered freshwater systems often relies on broad microbial community profiling using the 16S rRNA gene. Ensuring that data generated using such techniques accurately represent microbial community and nitrifying guild composition is essential for testing hypotheses regarding nitrifier distributions. The fourth goal of this thesis was to investigate how processing 16S rRNA gene amplicon sequencing data influences final reported relative abundances of nitrifiers. Processing five engineered freshwater datasets as merged paired-ends (“paired”), single-end using only the forward reads (“forward”), or single-end using only the reverse reads (“reverse”) revealed that the relative abundances of phylum Nitrospirota and genus Nitrospira were reduced when 16S rRNA gene amplicon sequencing data were processed as paired or reverse reads. When processed as forward reads, relative abundances of phylum Nitrospirota increased by as much as 21.9 times compared to paired, and detailed analysis of an aquarium biofilter dataset showed a 2.6- to 15.0-fold increase in genus Nitrospira relative abundances. Sequence analysis suggested that a higher frequency of chimera generation in reverse reads contaminates high quality forward reads when reads are merged during paired-end processing. These results serve as a warning for nitrification researchers to investigate their 16S rRNA gene amplicon sequencing datasets for potential Nitrospira-specific bias based on analysis artifacts.