NITROGEN CYCLE
Oxygen and nitrogen production by
an ammonia-oxidizing archaeon
Beate Kraft^1 *, Nico Jehmlich^2 , Morten Larsen^1 , Laura A. Bristow^1 , Martin Könneke3,4,
Bo Thamdrup^1 , Donald E. Canfield1,5,6
Ammonia-oxidizing archaea (AOA) are one of the most abundant groups of microbes in the
world’s oceans and are key players in the nitrogen cycle. Their energy metabolism—the oxidation of
ammonia to nitrite—requires oxygen. Nevertheless, AOA are abundant in environments where oxygen
is undetectable. By carrying out incubations for which oxygen concentrations were resolved to the
nanomolar range, we show that after oxygen depletion,Nitrosopumilus maritimusproduces dinitrogen
and oxygen, which is used for ammonia oxidation. The pathway is not completely resolved but likely
has nitric oxide and nitrous oxide as key intermediates.N. maritimusjoins a handful of organisms
known to produce oxygen in the dark. On the basis of this ability, we reevaluate the role ofN. maritimus
in oxygen-depleted marine environments.
A
mmonia-oxidizing archaea (AOA) are
only known to oxidize ammonia (NH 3 )
to nitrite (NO 2 −) using oxygen: NH 3 +
1.5O 2 →NO 2 −+H 2 O+H+( 1 , 2 ). Yet,
AOA are highly abundant in environ-
ments with very low, or even undetectable,
oxygen concentrations such as marine oxygen-
minimum zones (OMZs) and marine sediments
( 3 – 7 ). Certain populations of AOA become most
prevalent in oxygen-depleted zones of the water
column, indicating that they may be adapted
to these conditions ( 5 ).TheroleofAOAin
such environments is enigmatic, as they have
no known anaerobic metabolism. We used
trace luminescence oxygen sensors (here-
after, optodes) ( 8 ) to explore the physiology of
AOA at low nanomolar oxygen concentrations
and functional anoxia (oxygen levels below
detection) as typically found in OMZs ( 9 , 10 ),
thereby discovering oxygen production by
the marine AOANitrosopumilus maritimus
SCM1 ( 11 ). Dark, nonphotosynthetic oxygen
production is rare in nature, with only three
known pathways: chlorite dismutation during
perchlorate and chlorate respiration (ClO 2 −→
Cl−+O 2 ), detoxification of reactive oxygen
species (e.g., H 2 O 2 dismutation), and nitric
oxide (NO) dismutation (2NO 2 −→2NO→
N 2 +O 2 )( 12 ). Although the pathway of oxygen
production byN. maritimusis not fully re-
solved in this study, we show that it is not
any of the three aforementioned pathways.
The oxygen-producing pathway inN. maritimus
also involves NO dismutation but has oxygen
and nitrous oxide (N 2 O) as products and is thus
distinct from the NO dismutation described
above. Given the abundance ofN. maritimus
in oxygen-sparse environments, dark oxy-
gen production may be common in nature.
We also show that oxygen production is ac-
companied by N 2 production and thereby
identify a previously unknown and poten-
tially environmentally impactful N 2 produc-
tion pathway.
We first grew axenic cultures ofN. maritimus
aerobically as an ammonia oxidizer. The cul-
tures were then sparged with argon to oxygen
levels below 5mM, where the remaining oxygen
was consumed byN. maritimusthrough con-
tinued ammonia oxidation, indicating physio-
logically active cells. Unexpectedly, after oxygen
was completely consumed down to the limit
of detection of 1 nM, it immediately beganto slowly increase (Fig. 1A). A series of addi-
tions of oxygen-saturated water showed the
same recurring pattern: oxygen was consumed
on oxygen addition and increased directly
thereafter (Fig. 1A). In undisturbed incuba-
tions during which no oxygen additions were
made, oxygen accumulated over hours and
reached levels of between 100 and 200 nM
(Fig. 1A). This pattern was observed repro-
ducibly in multiple incubations carried out
over 2 years.
In comparison, no oxygen buildup was de-
tected in filtered abiotic controls or when cells
were killed by the addition of mercuric chloride
(fig. S1), ruling out the possibility of abiotic
oxygen production or intrusion of oxygen into
the incubation bottle. Contamination by
oxygen intrusion was further ruled out by
incubations in an anaerobic chamber, which
showed the same trend of oxygen production
(fig. S2). Involvement of medium components
(e.g., HEPES, EDTA) in oxygen production
was also excluded (fig. S3), and furthermore,
oxygen microelectrodes, which make use of
a different oxygen measurement principle,
showed the same patterns of oxygen increase
as did the optode measurements (fig. S4). In-
cubations withN. maritimusin medium con-
taining pyruvate showed no difference when
compared with incubations without pyru-
vate (fig. S5), thus ruling out oxygen produc-
tion by H 2 O 2 dismutation (H 2 O 2 →H 2 +O 2 )
because pyruvate reacts with and removes
H 2 O 2 via an abiotic decarboxylation reac-
tion ( 13 ).
As described in more detail below, we mea-
sured NO accumulation in our experiments
with a microelectrode and found a small NO
interference with the optode measurements
of O 2 (fig. S6) that we could calibrate. NO,
however, did not interfere with O 2 micro-
electrode measurements. When optode O 2SCIENCEscience.org 7 JANUARY 2022•VOL 375 ISSUE 6576 97
(^1) Nordcee, Department of Biology, University of Southern
Denmark, Odense, Denmark.^2 Department of Molecular
Systems Biology, Helmholtz Centre for Environmental
Research UFZ GmbH, Leipzig, Germany.^3 Marine Archaea
Group, Center for Marine Environmental Sciences (MARUM),
and Department of Geosciences, University of Bremen,
Bremen, Germany.^4 Institute for Chemistry and Biology of
the Marine Environment (ICBM), University of Oldenburg,
Oldenburg, Germany.^5 Key Laboratory of Petroleum
Geochemistry, Research Institute of Petroleum Exploration
and Development, China National Petroleum Corporation,
Beijing 100083, China.^6 Danish Institute of Advanced Study,
University of Southern Denmark, Odense, Denmark.
*Corresponding author. Email: [email protected]
Fig. 1. Oxygen production byN. maritimus.(A) After supplied oxygen is consumed, oxygen concentrations
immediately start to increase again (0 to 8 hours). When the incubation is left undisturbed and no
oxygenated water is added, the oxygen concentration increases over time (8 to 20 hours). (B) Cyanide
additions lead to a strong increase in oxygen production. Two parallel incubations showed the same
pattern as observed in (A): Oxygen increased immediately after added oxygen had been consumed
and accumulated over time when no oxygen additions were performed. After the additions of cyanide
(CN−, 0.5 mM final concentration; indicated with arrows), oxygen accumulations strongly increased.
Star symbols indicate additions of oxygen-saturated water. Black and gray lines represent the
two parallel incubations.
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