Enrichment and activity of comammox Nitrospira from the rotating biological contactors of a municipal wastewater treatment system

Abstract

Nitrification, the biological conversion of ammonia to nitrate, via nitrite, is an important process in both natural and engineered systems. Nitrification within wastewater treatment plants (WWTPs) is necessary because elevated ammonia concentrations are toxic to fish and can lead to eutrophication of receiving waters. Although the end product of nitrification (i.e., nitrate) also contributes to eutrophication, it is preferred over ammonia because nitrate has no direct oxygen demand and it is often converted to inert dinitrogen by denitrification. Nitrification is mediated by the combined activities of chemolithoautotrophic ammonia and nitrite oxidizers. The recent discovery of Nitrospira representatives capable of complete ammonia oxidation (comammox) to nitrate via nitrite has required that previously characterized systems be re-examined for potential contributions of comammox Nitrospira to nitrification. Although the abundance and diversity of comammox Nitrospira in the rotating biological contactors (RBCs) of a municipal WWTP in Guelph Ontario were recently examined using several cultivation-independent approaches, little is known about the activity of comammox Nitrospira in these biofilm-based systems. Additionally, lack of a suitable comammox Nitrospira enrichment culture impedes future efforts to explore the biochemical and physiological characteristics of these WWTP nitrifiers. The aim of this thesis research was to enrich comammox Nitrospira from Guelph RBCs and assess their contributions to nitrification within the WWTP biofilm. The enrichment cultures were prepared using a combination of differential size filtration and antimicrobial supplementation. The growth and activity of resulting enrichment cultures were examined using ammonia depletion and nitrate production, as well as end-point PCR targeting the amoA genes coding for subunit A of the ammonia monooxygenase of comammox Nitrospira, ammonia-oxidizing bacteria (AOB), and ammonia-oxidizing archaea (AOA). The amoA-based phylogeny reveals that the two comammox bacteria grown in enrichment cultures are phylogenetically distinct and represent novel ecotypes, both belonging to clade A sublineage II Nitrospira. Culture G4 is an enrichment that contains novel AOA and comammox Nitrospira species. In addition to possessing the genetic machinery for complete ammonia oxidation, the comammox Nitrospira enriched in G4 also possesses a gene that codes for a cyanase (cynS gene). The presence of a cyanase may allow these comammox bacteria to breakdown cyanate to ammonium in ammonia-limited environments. Preliminary results showed that G4 grew with ammonium (from cyanate breakdown), but it remains unclear whether these comammox Nitrospira were able to breakdown cyanate biotically using cyanase. The enrichment culture G8 contains a comammox Nitrospira representative that is phylogenetically distinct from known comammox Nitrospira representatives. Activity assays demonstrated that both enrichment cultures (G4 and G8) grew under ammonia-fed conditions, with complete conversion of ammonia to nitrate. Enrichment cultures will help to further explore physiological characteristics, such as ammonia and nitrite affinities, as well as alternative metabolisms of comammox Nitrospira in the RBCs. In addition to cultivation efforts, my research explored the potential contributions of nitrifiers to RBC biofilm nitrification. Microcosm incubations were established to test the effects of five nitrification inhibitors (i.e., c-PTIO, ATU, DCD, chlorite, and chlorate) on biofilm samples. Additionally, the effects of c-PTIO, ATU, DCD, and simvastatin were investigated on comammox Nitrospira enrichment cultures. Results from enrichment culture incubations indicated that comammox Nitrospira are inhibited by 10 µM ATU and 10 mM DCD and are insensitive to 100 µM c-PTIO and 8 µM simvastatin. In RBC biofilm suspensions, preliminary evidence suggests that comammox Nitrospira were inhibited by ATU, DCD, chlorite, and possibly chlorate, suggesting that they are likely active members of the nitrifying community in the RBCs. The results of this thesis underline the complexity of the nitrifying community in the RBC biofilm and highlight the need for including comammox Nitrospira when evaluating contributions of different nitrifiers to ammonia oxidation. Multiple “omic” approaches, in conjunction with activity assays, will likely be valuable for examining the metabolic versatility and activity of comammox Nitrospira, as well as other nitrifiers, in the RBCs. Finally, work presented in this thesis opens new research avenues to explore with enrichment cultures to better characterize the potential metabolic roles for comammox Nitrospira in the RBCs, as well as other engineered and natural aquatic environments.