The Unknown Bacteria Behind Anammox

Anammox, also known as anaerobic ammonium oxidation, is an emerging alternative to conventional nitrogen removal in wastewater treatment. The microorganisms that perform it live in a reactor where they convert water’s ammonium and nitrite into nitrogen gas anaerobically, without the energy-consuming aeration required by conventional methods.

Beyond its energy benefits, little is known about how anammox works exactly. One of the world’s first anammox reactors was installed at a French fries factory in Olburgen, The Netherlands in 2006. This reactor performed so well that its biomass was used to seed the anammox process at other wastewater treatment plants around the world, including the world’s largest in China.

After nearly a decade of service, researchers wanted a closer look at what was happening in that reactor.

“To really understand the physiology of the anammox bacteria, they should be studied under laboratory conditions, but their interaction with other organisms is important out in the environment,” said Daan Speth, a researcher from the department of microbiology at Radboud University in Njimegan, The Netherlands. “An anammox reactor like the one in Olburgen provides a great system to study anammox ‘in the wild’ while still having a stable and controlled system to work with.”

A research team from the BE-Basic Foundation and Radboud University, including Speth, found 23 near-complete draft genomes in the reactor, of which 19 have no known close, in some cases even distant, genomic relatives.

“[This research] shows that, even in a modern, man-made system, there is still an enormous amount of things to discover,” said Speth. “We have only begun to scratch the surface of the diversity that exists in nature.”

While the study is warranted for its microbiological merits alone, the insight it provides into the anammox process could further improve a wastewater nutrient removal technique that seems poised to become widespread in the industry.

For the anammox process to work, organisms known as AOB, or ammonium oxidizing bacteria, must work in tandem with the anammox bacteria. The research demonstrated that a substantial fraction of the AOB was present in places anammox was missing. If the operators could improve on this ratio by bringing the two together, they might increase reactor performance.

“Additionally, there are a lot of bacteria growing on organic carbon in the system,” said Speth. “Understanding their role and interaction with anammox and AOB will help improve the process for waste streams with different carbon content.”

The ascension of anammox has outpaced researchers’ ability to identify its nuts and bolts until recent advances in genome science made the work of Speth and his team possible.

The researchers used a newfound process called genome-resolved metagenomics to conduct their study. They pretreated samples from the reactor to enrich or deplete certain microorganisms and find which DNA sequences remained abundant or subsided, allowing them to group similar ones together, or in some cases, find they did not fit in any previously known genome group.

“Our genome reconstruction method has only really recently become feasible for moderately complex systems like this reactor,” said Speth. “DNA sequencing chemistry and tools for analysis have greatly improved over the past years, to the extent that our study would have been nearly impossible a few years ago.”

The study, published in Nature Communications under the title “Genome-based microbial ecology of anammox granules in a full-scale wastewater treatment system,” turned up a genome so mysterious that researchers are calling it “microbial dark matter.”

“Within microbial dark matter, there is a group of organisms that look like bacteria, but they’re missing a lot of essential genes,” said Speth. “These organisms might be bacterial parasites, we don’t know yet. In our data, there are five [such] genomes, although they were not very abundant. If there are parasites, studying their interaction with other bacteria might help improve the community [inside the anammox reactor].”

As its secrets are revealed anammox stands to receive wider acceptance from the wastewater treatment industry.