Oxidation kinetics and inverse isotope effect of marine nitrite-oxidizing isolates

Abstract

Nitrification, the step-wise oxidation of ammonium to nitrite and nitrate, is important in the marine environment because it produces nitrate, the most abundant marine dissolved inorganic nitrogen (DIN) component and N-source for phytoplankton and microbes. This study focused on the second step of nitrification, which is carried out by a distinct group of organisms, nitrite-oxidizing bacteria (NOB). The growth of NOB is characterized by nitrite oxidation kinetics, which we investigated for 4 pure cultures of marine NOB (Nitrospina watsonii 347, Nitrospira sp. Ecomares 2.1, Nitrococcus mobilis 231, and Nitrobacter sp. 311). We further compared the kinetics to those of non-marine species because substrate concentrations in marine environments are comparatively low, which likely influences kinetics and highlights the importance of this study. We also determined the isotope effect during nitrite oxidation of a pure culture of Nitrospina (Nitrospina watsonii 347) belonging to one of the most abundant marine NOB genera, and for a Nitrospira strain (Nitrospira sp. Ecomares 2.1). The enzyme kinetics of nitrite oxidation, described by Michaelis-Menten kinetics, of 4 marine genera are rather narrow and fall in the low end of halfsaturation constant (K m ) values reported so far, which span over 3 orders of magnitude between 9 and >1000 μM NO 2 − . Nitrospina has the lowest K m (19 μM NO 2 − ), followed by Nitrobacter (28 μM NO 2 − ), Nitrospira (54 μM NO 2 − ), and Nitrococcus (120 μM NO 2 − ). The isotope effects during nitrite oxidation by Nitrospina watsonii 347 and Nitrospira sp. Ecomares 2.1 were 9.7 ± 0.8 and 10.2 ± 0.9 ‰, respectively. This confirms the inverse isotope effect of NOB described in other studies; however, it is at the lower end of reported isotope effects. We speculate that differences in isotope effects reflect distinct nitrite oxidoreductase (NXR) enzyme orientations.

Genomic and kinetic analysis of novel Nitrospinae enriched by cell sorting

“…= 2.1, n = 8) and the maximum reaction rate, V max , to be 41.4 µmol NO 2 − mg protein −1 h −1 (s.d. = 9.4, n = 6), which is highly similar to the previously reported K m(app) and V max of the closely related N. watsonii (18.7 ± 2.1 µM NO 2 − and 36.8 µmol NO 2 − mg protein −1 h −1 ) [ 55 ]. The K m(app) measured for “ Ca .…”

“…Nitronauta litoralis” EB; n = 8 for N. gracilis ). The other K m(app) values were retrieved from previous studies [ 54 , 55 , 81 ]. …”

Niche Differentiation of Host-Associated Pelagic Microbes and Their Potential Contribution to Biogeochemical Cycling in Artificially Warmed Lakes

“…We identified two OTUs that belonged to the genera of Nitrobacter (class: alphaproteobacteria) which linked to N-cycling. Nitrobacter is responsible for the conversion of nitrite to nitrate which is an important pathway for nitrification (Mellbye et al, 2015;Daims et al, 2016;Jacob et al, 2017). A recent metagenomic study on zooplanktonassociated bacterial communities in marine environment detected several genes (e.g., nar, nor, nos) which could be responsible for N-cycling, especially, nitrification, denitrification and dissimilatory nitrate, and nitrite reduction to ammonium (DNRA) (De Corte et al, 2018).…”

Nitrifier adaptation to low energy flux controls inventory of reduced nitrogen in the dark ocean

“…These observations suggest that marine nitrite-oxidizing bacteria (NOB), among which populations of Nitrospina and Nitrospira are most ubiquitous (11)(12)(13)(14), have similarly high affinities for nitrite. However, cultured representatives of NOB mainly originate from nonoxygenated oceanic environments, and their K m values are orders of magnitude higher than those of marine AOA (10,(15)(16)(17)(18)(19)(20)(21)(22). Only one recent study showed that the half-saturation constant K s (254 ± 161 nM) for nitrite oxidation is of the same magnitude as that of marine AOA in the low-oxygen seawater of the Eastern…”