Study Notes on Autotrophic Ammonia-Oxidizing Marine Archaeon

Martin Koenneke1†, Anne E. Bernhard1†, José R. de la Torre1*, Christopher B. Walker1, John B. Waterbury2 & David A. Stahl1
1Department of Civil and Environmental Engineering, University of Washington, Seattle, Washington 98195, USA.
2Woods Hole Oceanographic Institute, Woods Hole, Massachusetts 02543, USA.
*Present addresses: Institut für Chemie und Biologie des Meeres, Universität Oldenburg, Oldenburg 26111, Germany (M.K.); Department of Biology, Connecticut College, New London, Connecticut 06320, USA (A.E.B.).

Microbiologists historically characterized Archaea as obligate extremophiles that thrive in harsh environments.
Limited physiological diversity among cultivated Archaea suggested a metabolic constraint to specific niches, e.g., cultivated Crenarchaeota being primarily sulphur-metabolizing thermophiles.
Landmark studies using cultivation-independent methods revealed vast numbers of Crenarchaeota in cold oxic ocean waters, indicating their ubiquity in aquatic and terrestrial environments.
Approximate dominance of marine Crenarchaeota in oceans estimated at $10^{28}$ cells, indicating their significant role in global biogeochemical cycles.
Isotopic analyses of marine crenarchaeal lipids suggested these planktonic Archaea can fix inorganic carbon.

Isolation of a marine crenarchaeote, which grows chemolithoautotrophically by aerobically oxidizing ammonia to nitrite.
This detection marks the first observation of nitrification in Archaea.
The autotrophic metabolism of the isolated organism and its close phylogenetic relationship to environmental samples suggest it may significantly impact global carbon and nitrogen cycles.

Marine Crenarchaeota recognized as a dominant fraction of ocean bacterioplankton, comprising up to 40% of the bacterioplankton in deep ocean waters.
Previous studies indicated potential autotrophy among untapped natural populations, and significant ammonium uptake was noted.
Availability of pure culture will advance studies regarding their physiology, evolutionary origins, and contributions to oceanic biogeochemical cycles.

Known nitrifying bacteria categorized into two groups:
- Those that oxidize ammonia to nitrite.
- Those that oxidize nitrite to nitrate.
No existing genera have been linked to the complete oxidation of ammonia to nitrate.

Initial suspicion of archaeal involvement in nitrification derived from ribosomal RNA gene surveys.
Detection of sequences linked to marine group 1 Crenarchaeota in various nitrifying environments (e.g., Plum Island Sound, Shedd Aquarium).
Cultures enriched in Crenarchaeota were obtained by supplementing filtered aquarium water with ammonium chloride and inoculating with specific gravel samples.

Cultures enriched for Crenarchaeota were incubated in the dark at temperatures of 21-23 °C.
The culture was enriched to approximately 90% Crenarchaeota after six months.
Oxidation of ammonia to nitrite was strongly linked to augmented levels of Crenarchaeota, validated via quantitative PCR and observational data.

Defined media used for the isolation of Crenarchaeota included bicarbonate and ammonia as carbon and energy sources.
Pure culture designated SCM1 achieved confirmation of purity through multiple methods including quantitative PCR and fluorescent in situ hybridization (FISH).
Amplification yielded 16S rRNA gene sequences, revealing high levels of identity (.98%) with marine group 1 Crenarchaeota from various geographical locations.

Members of marine group 1 Crenarchaeota demonstrated only 84% sequence identity with low-temperature soil Crenarchaeota and less than 80% with cultivated thermophilic Crenarchaeota.
Phylogenetic analyses indicate that low-temperature Crenarchaeota are more closely related to each other than to thermophilic relatives.

Cells visualized via electron microscopy appear as straight rods, measuring 0.17–0.22 mm in diameter and 0.5–0.9 mm in length.
Cells exhibited no apparent flagella or cellular compartments and displayed a peanut-like shape upon staining.
Maximum cell density of SCM1 reached $1.4 imes 10^7$ cells/ml at 28 °C with minimum generation time of 21 hours.

Ammonium concentrations in oceanic and coastal environments range significantly, which may influence marine Crenarchaeota growth.
SCM1’s maximum growth rate (0.78 d^{-1}) exceeded natural bacterioplankton growth rates (0.05-0.3 d^{-1}).
Organic compounds inhibited growth, suggesting that excretions from other organisms might limit Crenarchaeota abundance in the marine ecosystem.
Growth correlated with near-stoichiometric conversion of ammonia to nitrite.

Ammonia monooxygenase (AMO) is responsible for the oxidation of ammonia to hydroxylamine, which is then converted to nitrite by hydroxylamine oxidoreductase.
The reaction can be mathematically represented as:
$NH3 + 1.5 O2 ightarrow NO2^{-} + H2O + H^+ \ (DG^0 = -235 kJ mol^{-1})$ .

AMO-related genes discovered in environmental sequences hint at widespread nitrifying potential in marine Crenarchaeota.
Comparisons of AMO-encoding genes from SCM1 with sequences from Sargasso Sea and soil Crenarchaeota revealed significant amino acid similarity (93-98% and 80-90% respectively).
The study firmly correlates archaea’s AMO genes with nitrification activity.

The study establishes "Nitrosopumilales" order nov. and "Nitrosopumilaceae" family nov.
"Nitrosopumilus maritimus" designated as the new genus and species.
Etymology:
- "nitrosus" (nitrous),
- "pumilus" (dwarf),
- "maritimus" (of the sea).

The organism's chemolithoautotrophy enables survival in oligotrophic environments, potentially contributing to primary productivity without organic energy sources.
Further investigation is needed to elucidate pathways pertaining to mesophilic Crenarchaeota.
The discovery raises questions regarding the evolutionary origins of ammonia oxidation and the ancestral characteristics of nitrifiers.

The isolation of a nitrifying archaeon signifies a groundbreaking moment in understanding archaeal ecology and potential contributions to biogeochemical cycles.

Cultures grown in Synthetic Crenarchaeota Media at 28 °C.
PCR amplification and cloning of 16S rRNA gene sequences were performed for phylogenetic analysis.
Various microscopy techniques (transmission and scanning electron microscopy) were utilized for detailed morphological observations.
Fluorescence techniques helped in determining cell populations and characteristics based on 16S rRNA hybridization.

Acknowledgments for technical assistance and resources, particularly the Shedd and Seattle aquariums, supported by National Science Foundation programs.

Extensive list of references showing prior studies and genetic analysis methods, indicating a strong research foundation that underpins this study's results.