Nitrous Oxide Reduction Kinetics Distinguish Bacteria Harboring Clade I NosZ from Those Harboring Clade II NosZ

Abstract

Nitrous oxide (N 2 O) is a potent greenhouse gas with a strong potential to drive climate change (1, 2) and will continue to be the largest contributor to ozone depletion in the stratosphere (2, 3). Anthropogenic activities, predominantly, fertilizer application in agricultural production, have contributed to a steady increase in atmospheric N 2 O concentrations, and a continued upward trend is expected (4-6). Particularly troublesome are the findings of a recent study that concluded that without solutions for the N 2 O problem, carbon dioxide (CO 2 ) emission reductions even greater than those already proposed will be required to avoid climate change (7). Due to its environmental impact, the pathways leading to the generation and consumption of N 2 O have received heightened interest.In the environment, N 2 O is predominantly formed as an intermediate of denitrification and a nitrification by-product (8). Denitrification is the stepwise reduction of NO 3 Ϫ /NO 2 Ϫ to gaseous products (i.e., N 2 O, N 2 ), with each step being mediated by distinct enzyme systems (9). A kinetic imbalance in the rates of reactions producing and consuming N 2 O during denitrification leads to the release of N 2 O to the atmosphere (8, 10). In nitrification, N 2 O is generated by nitrifier denitrification and as a byproduct of ammonia oxidation (8,11,12). A recent report indicated that nitrifiers, rather than denitrifiers, may be the primary source of N 2 O in agricultural soils (12). Other processes contributing to N 2 O formation include respiratory ammonification (also known as dissimilatory nitrate/nitrite reduction to ammonium [DNRA]) and chemodenitrification (i.e., the abiotic reaction of NO 2 Ϫ with ferrous iron) (13,14). In contrast to the diverse pathways of N 2 O generation, the only known major biological pathway for the removal of N 2 O is by reduction to N 2 , catalyzed by the enzyme nitrous oxide reductase (NosZ) (8,(15)(16)(17)

Nitrous Oxide Reduction by an Obligate Aerobic Bacterium, Gemmatimonas aurantiaca Strain T-27

“…Thus, these nondenitrifying N 2 O reducers function as de facto sinks of N 2 O, as confirmed by physiological observations in experiments with isolates possessing NosZ (20,21). Indeed, a recent study on the kinetics of N 2 O reductions revealed that the clade II nosZ-containing organisms have significantly higher affinity to N 2 O than the clade I nosZ-containing organisms, supporting the hypothesis that the organisms with clade II nosZ may contribute to the mitigation of N 2 O emissions from nonpoint sources (17).…”

“…In P. denitrificans, the high level of nosZ transcription was sustained in the anoxic phase until all nitrogen oxides were depleted, indicating that NosZ enzymes synthesized during anoxic incubation were utilized for N 2 O reduction (52). Further, in four strains of bacteria examined for kinetic properties, N 2 O served as the sole source of electron acceptor for exponential growth in the complete absence of O 2 (17). As nitrous oxide reductase is active only in the absence of O 2 and thus is supposedly more beneficial for its owners under anoxic conditions (52), the disappearance of nosZ transcription activity in oxygen-depleted cultures of G. aurantiaca strain T-27 is rather unexpected and is unprecedented in any studies on denitrifiers or nondenitrifying N 2 O reducers alike.…”

“…The amounts of N 2 O in the serum bottles were quantified using an HP6890 series gas chromatograph equipped with an HP-PLOT/Q column and an electron capture detector (Agilent Technologies, Santa Clara, CA). The injector, oven, and detector temperatures were set to 200, 85, and 250°C, respectively (17). For each measurement, 200 l of headspace gas was removed using a 1700 series gas-tight syringe (Hamilton Company, Reno, NV), and 100 l of the withdrawn sample was manually injected into the gas chromatograph.…”

“…Oxygen concentrations were monitored with fiber-optic oxygen sensor spots and a FireStingO2 oxygen meter (Pyroscience, Aachen, Germany). The total amounts of N 2 O and O 2 in the reaction vessels were calculated from the headspace concentrations as described previously (17). The dimensionless Henry's constants (calculated as moles in the headspace/ moles in the aqueous phase) of N 2 O and O 2 at 30°C were calculated to be 1.92 and 33.3, respectively (61).…”

“…Other relatively minor sources of N 2 O include dissimilatory reduction to ammonium (DNRA) and chemodenitrification (8,(12)(13)(14). In contrast to the diverse pathways leading to the production of N 2 O, the sole biological sink process of N 2 O in the environment is its reduction by the organisms expressing nitrous oxide reductases (NosZ) (8,(15)(16)(17). N 2 O reduction was originally regarded merely as a part of the denitrification cascade.…”