Meet the Microbes That Breathe Nitrate Instead of Oxygen – And They’re Everywhere

Scientists have discovered a remarkable new form of symbiosis — a bacterium that lives inside a single-celled organism (a ciliate) and provides it with energy. Unlike mitochondria, which use oxygen, this microbe powers its host by breathing nitrate.

Initially found in a freshwater lake, researchers set out to determine how widespread these microbes are. To their surprise, they uncovered them in diverse environments worldwide, from lakes and groundwater to even wastewater. This discovery challenges our understanding of microbial partnerships and reveals how these tiny organisms play a hidden yet significant role in global ecosystems.

A New Symbiotic Discovery

In 2021, scientists at the Max Planck Institute for Marine Microbiology in Bremen, Germany, made a remarkable discovery: a unique bacterium that lives inside a ciliate — a single-celled eukaryote — and provides it with energy. This symbiotic relationship is similar to the role mitochondria play in cells, but with one major difference: instead of using oxygen, this endosymbiont generates energy by respiring nitrate.

To better understand the distribution and diversity of these unusual microbes, the researchers in Bremen expanded their study. Now the researchers from Bremen set out to learn more about the environmental distribution and diversity of these peculiar symbionts. “After our initial discovery of this symbiont in a freshwater lake, we wondered how common these organisms are in nature,” explains Jana Milucka from the Max Planck Institute for Marine Microbiology. “Are they extremely rare and therefore eluded detection so long? Or do they exist elsewhere and if so, what are their metabolic capacities?”

A Global Inhabitant

To find answers, the scientists searched massive public sequencing databases containing genetic data from a wide range of environmental samples. Their findings were surprising: these symbionts appeared in about 1,000 different datasets. “We were surprised how ubiquitous they are. We could find them on every inhabited continent,” says Milucka. “Moreover, we learned that they can live not only in lakes and other freshwater habitats but also in groundwater and even wastewater.”

Meet the Family: New Members Do New Tricks

The scientists discovered not only the original symbiont in these datasets, but also some new close relatives. “We ended up identifying four new species, two of which actually constituted a new genus. Because this new genus of symbionts likely has a similar role as the originally discovered Azoamicus (name meaning “nitrogen friend”), we named the new genus Azosocius (“nitrogen associate”), explains first-author Daan Speth. “Lucky for us, one of the new Azosocius species was retrieved not too far from Bremen, from a groundwater sample in Hainich, Germany.”

Evolving Capabilities: A Surprising Oxygen Connection

Now the scientists wanted to dig deeper into the life of these new species. Thanks to a collaboration with Kirsten Küsel and Will Overholt from the Friedrich Schiller University in Jena, Germany, who initially collected the Hainich samples, they were able to access the sampling site and look into metatranscriptomic data, i.e. data describing the gene expression in the sample and indicating microbial activity.

“Here, we were in for another surprise – these respiratory symbionts can do new tricks,” Speth continues. Unlike the original symbiont species, which can only perform anaerobic respiration (i.e. denitrification), all new symbiont species actually encode a terminal oxidase – an enzyme that enables them to also respire oxygen in addition to nitrogen. “This can explain why we find these symbionts also in environments that are fully or partially oxic.”

Evolutionary and Ecological Implications

These results, now presented in the journal Nature Communications, answer the scientists’ open questions regarding the symbiont’s biogeography. “Thanks to the discovery of these new species, we can now also start thinking more about their evolution,” Milucka looks ahead. “We can hopefully understand better how these beneficial symbioses begin and how they evolve over time.”

Moreover, there is an ecological aspect to this research: “By performing denitrification, this symbiosis impacts the nitrogen cycle of their respective habitat and has the potential to remove nutrients, such as nitrogen oxides, as well as produce greenhouse gases, such as nitrous oxide,” adds Speth.

Marvels of Microbial Symbiosis

And last but not least, there is the simple appreciation of the intriguing world of microbes. “This organism is a marvel of nature,” Milucka enthuses. “Protists are capable of such astonishing metabolic innovations, often because they so readily jump into relationships with prokaryotes. To me, this is just fascinating. When it comes to understanding the evolution of eukaryotes, these organisms are an important piece of the puzzle.”