Genotypic and phenotypic characterization of hydrogenotrophic denitrifiers

Abstract

Stimulating litho-autotrophic denitrification in aquifers with hydrogen is a promising strategy to remove excess NO 3À , but it often entails accumulation of the cytotoxic intermediate NO 2 À and the greenhouse gas N 2 O. To explore if these high NO 2 À and N 2 O concentrations are caused by differences in the genomic composition, the regulation of gene transcription or the kinetics of the reductases involved, we isolated hydrogenotrophic denitrifiers from a polluted aquifer, performed whole-genome sequencing and investigated their phenotypes. We therefore assessed the kinetics of NO 2 À , NO, N 2 O, N 2 and O 2 as they depleted O 2 and transitioned to denitrification with NO 3 À as the only electron acceptor and hydrogen as the electron donor. Isolates with a complete denitrification pathway, although differing intermediate accumulation, were closely related to Dechloromonas denitrificans, Ferribacterium limneticum or Hydrogenophaga taeniospiralis. High NO 2 À accumulation was associated with the reductases' kinetics. While available, electrons only flowed towards NO 3 À in the narG-containing H. taeniospiralis but flowed concurrently to all denitrification intermediates in the napAcontaining D. denitrificans and F. limneticum. The denitrification regulator RegAB, present in the napA strains, may further secure low intermediate accumulation. High N 2 O accumulation only occurred during the transition to denitrification and is thus likely caused by delayed N 2 O reductase expression.

pH-Dependent Hydrogenotrophic Denitratation Based on Self-Alkalization

“…It is possible that a strong preference for electron flow from a common pool of reductant to NarG could prevent other nitrogen oxide reductases from receiving electrons as long as nitrate is available, thus causing a substantial nitrite accumulation like that observed in previous studies. 55,60,61 Instead, genotypes with NapA only were found to reduce nitrate to dinitrogen gas without nitrite accumulation, suggesting no favorable competition for electrons with downstream reductases. 60,61 Although the primary, active members in our study (e.g., co.bin.3 and H2-1.bin.15) possessed NapA, we did not find significant nitrite reduction whether nitrate was present or not (Figure S3), negating the hypothesis of electron competition by the relevant enzymes.…”

“…55,60,61 Instead, genotypes with NapA only were found to reduce nitrate to dinitrogen gas without nitrite accumulation, suggesting no favorable competition for electrons with downstream reductases. 60,61 Although the primary, active members in our study (e.g., co.bin.3 and H2-1.bin.15) possessed NapA, we did not find significant nitrite reduction whether nitrate was present or not (Figure S3), negating the hypothesis of electron competition by the relevant enzymes. In the case here with high pH, protons outside of cells were likely deficient, resulting in the periplasmic reductases (e.g., NapA, NirS, and NirK) lacking the protons required for turnover.…”

How to Provide Nitrite Robustly for Anaerobic Ammonium Oxidation in Mainstream Nitrogen Removal

“…To achieve complete autotrophic nitrogen removal, the coexistence pattern of these autotrophic microbes requires deeper study, i.e., it requires a determination of the coexistence pattern of autotrophic denitrifiers with AnAOB, such as granulation, biofilm development, and gel-encapsulation of bacterial biomass, for achieving satisfactory biomass retention . Simultaneously, the mechanism of nitrite accumulation in autotrophic nitrate reduction is still unclear, e.g., whether autotrophic denitrifiers possess an absence of genes encoding the denitrification reductases, and whether the transcriptional regulation and post-translational phenomena of these reductases could be determined by environmental conditions. ,, …”

“…Autotrophic PD is depended on the electron donors such as sulfur, hydrogen, iron and electricity. ,− It seems to have an advantage over heterotrophic PD when incorporated into the anammox process for its advantages of high efficiency, economy, low GHG emissions, and safety to AnAOB . Typically, dosing chemicals (such as Na 2 S 2 O 3 and Fe­(II)) and establishing bioelectrochemical systems are common ways to integrate autotrophic nitrate reduction with the anammox process, while creating an environment favoring these autotrophic microbes, i.e., AnAOB, sulfur-oxidizing bacteria, hydrogenotrophic denitrifiers, iron-reducing bacteria, and electroactive bacteria.…”

Denitrification by bradyrhizobia under feast and famine and the role of the bc1 complex in securing electrons for N2O reduction

“…One example is denitrification, which is the reduction of NO 3 − to N 2 through anaerobic respiration where the N-oxides are used as terminal electron acceptors when O 2 becomes scarce. This process can be performed by a diverse range of heterotrophic bacteria, archaea and fungi, which use various forms of organic compounds as electron donors to obtain energy (6) or, in some cases, H 2 (7). The last step of denitrification is the reduction of N 2 O, a strong climate gas, to harmless N 2 , catalyzed by the N 2 O reductase (Nos) (8,9).…”