Abstract
Nitrite-oxidizing bacteria (NOB) catalyze the second step of nitrification, which is an important process of the biogeochemical nitrogen cycle and is exploited extensively as a biological nitrogen removal process. Members of the genus Nitrospira are often identified as the dominant NOB in a diverse range of natural and artificial environments. Additionally, a number of studies examining the distribution, abundance, and characterization of complete ammonia oxidation (comammox) Nitrospira support the ecological importance of the genus Nitrospira. However, niche differentiation between nitrite-oxidizing Nitrospira and comammox Nitrospira remains unknown due to a lack of pure cultures. In this study, we report the isolation, physiology, and genome of a novel nitrite-oxidizing Nitrospira strain isolated from a fixed-bed column at a drinking water treatment plant. Continuous feeding of ammonia led to the enrichment of Nitrospira-like cells, as well as members of ammonia-oxidizing genus Nitrosomonas. Subsequently, a microcolony sorting technique was used to isolate a novel nitrite-oxidizing Nitrospira strain. Sequences of strains showing the growth of microcolonies in microtiter plates were checked. Consequently, the most abundant operational taxonomic unit (OTU) exhibited high sequence similarity with Nitrospira japonica (98%) at the 16S rRNA gene level. The two other Nitrospira OTUs shared over 99% sequence similarities with N. japonica and Nitrospira sp. strain GC86. Only one strain identified as Nitrospira was successfully subcultivated and designated as Nitrospira sp. strain KM1 with high sequence similarity with N. japonica (98%). The half saturation constant for nitrite and the maximum nitrite oxidation rate of strain KM1 were orders of magnitude lower than the published data of other known Nitrospira strains; moreover, strain KM1 was more sensitive to free ammonia compared with previously isolated Nitrospira strains. Therefore, the new Nitrospira strain appears to be better adapted to oligotrophic environments compared with other known non-marine nitrite oxidizers. The complete genome of strain KM1 was 4,509,223 bp in length and contained 4,318 predicted coding sequences. Average nucleotide identities between strain KM1 and known cultured Nitrospira genome sequences are 76.7–78.4%, suggesting at least species-level novelty of the strain in the Nitrospira lineage II. These findings broaden knowledge of the ecophysiological diversity of nitrite-oxidizing Nitrospira.
Highlights
Nitrification is a key process in the biogeochemical nitrogen cycle
The ammonia concentration in the bioreactor temporarily increased around the 300 days, the nitrate concentration increased with an increase in the influent ammonia concentration
The ratios of species belonging to the Nitrosomonas and Nitrospira lineage II on day 350 were around 30 and 40%, respectively, based on the cell counts
Summary
Nitrification is a key process in the biogeochemical nitrogen cycle This reaction takes place in two-steps: the first step is ammonia oxidation, which is carried out by ammoniaoxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB), and the second step is nitrite oxidation, which is carried out by the nitrite-oxidizing bacteria (NOB). It has been reported that a group of Nitrospira species that are conventionally recognized as NOB, carry out complete ammonia oxidation (comammox), transforming ammonia to nitrate (Daims et al, 2015; van Kessel et al, 2015). This surprising finding suggests the ecological importance of Nitrospira and indicates that our understanding about nitrification is still insufficient. Recent metagenomic analysis revealed the presence of comammox Nitrospira in activated sludge (Chao et al, 2016; Camejo et al, 2017), soils (Orellana et al, 2018; Shi et al, 2018), freshwater (Liu et al, 2020), and drinking water treatment plants (DWTPs) (Pinto et al, 2015; Wang et al, 2017)
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