Tokyo Institute of Technology and RIKEN discover artificial catalyst without oxygen molecules — Anaerobic activation of ammonia

A research team led by Professor Ryuhei Nakamura and Researcher He Daoping of the Earth-Life Science Institute at Tokyo Institute of Technology, along with Team Leader Daisuke Hashizume and Technical Staff Kiyohiro Adachi of RIKEN, discovered that copper sulfide (covellite: CuS), which is a mineral that exists in anaerobic environments, can artificially drive anaerobic ammonia oxidation (anammox) performed by bacteria. By analyzing the reaction mechanism, the research team confirmed that copper sulfide can reproduce the entire biological anammox pathway composed of three metalloenzymes. The results are expected to lead to the development of a new wastewater treatment technology and were published in Nature Chemistry on May 24.

Anaerobic ammonia oxidation (anammox), a reaction that generates nitrogen gas from ammonia and nitrite, was discovered in 1995 in bacteria found in wastewater treatment plants. Until then, it was believed that molecular oxygen is necessary for the activation of ammonium ions. The anammox reaction is catalyzed by three metalloenzymes (nitrite reductase, hydrazine synthase, and hydrazine dehydrogenase) present in an organelle called the anammoxosome in a bacterial cell. It is recognized as an important biological process as well as a denitrification reaction, contributing to the removal of up to 50% of fixed nitrogen present in the ocean. It is expected to be an efficient and sustainable method that does not require aerobic conditions, unlike conventional wastewater treatment processes. Meanwhile, no artificial catalyst that can catalyze anammox reactions has been found.

In this study, the research group investigated the catalytic activity of 37 types of metals, metal sulfides, metal oxides, and metal hydroxides to identify nonenzymatic catalysts that drive the anammox reaction. Specifically, isotope-labeled ¹⁴NO₂⁻ and ¹⁵NH₄⁺ were added as substrates to phosphate buffer at pH 7 to culture anammox microbes, and substances produced were analyzed via gas chromatography−mass spectrometry. As a result, they discovered that copper sulfide (CuS) is an artificial catalyst that activates ammonia without oxygen molecules. X-ray diffraction and Raman spectroscopy also revealed that synthetic CuS, which exhibited catalytic activity for the anammox reaction, has the same structure as naturally occurring covellite minerals.

The most characteristic reaction step of the anammox reaction is the synthesis of hydrazine, which is the most powerful reducing agent in nature and has been used in industrial applications including propellants for rocket engines. Therefore, verification of whether hydrazine is formed in the anammox reaction with CuS was undertaken and the group succeeded in detecting a hydrazine intermediate. It was also confirmed that hydrazine generated by the CuS catalyst was oxidized to nitrogen molecules, as in the in vivo reaction. Furthermore, they succeeded in reproducing the anammox reaction, which is catalyzed by three enzymes in biological systems, with the catalytic action of a single covellite (CuS) mineral. Because copper sulfide (CuS) catalysts occur naturally as covellite minerals, mineral-driven ammonia activation and hydrazine synthesis are also expected to contribute to origin-of-life research, where the synthesis of nitrogen-containing biomolecules such as amino acids and nucleobases is the key.

Nakamura said, "We would like to develop a new water purification technology based on this result by substantially increasing the activity of the catalyst. We would also like to explore the origin of nitrogen metabolism on the primitive Earth based on the fact that we were able to reconstruct enzymatic reactions with minerals."

Journal Information
Publication: Nature Chemistry
Title: Copper sulfide mineral performs non-enzymatic anaerobic ammonium oxidation through a hydrazine intermediate
DOI: 10.1038/s41557-024-01537-6