David Stahl's postdoc Anne Bernhard hunts for Archaea samples at the Plum Island estuary.

The hunt for conclusive evidence that Archaea turn ammonia into nitrites took David Stahl's research team more than 11 years. The search began with water samples from Chicago's Shedd Aquarium, led to a New England estuary, then to the Seattle Aquarium, and concluded at the lab bench with a rigorous process of purification and characterization, before turning into a paper (see page 543).

Stahl had been interested in marine nitrification of ammonia for about 15 years and knew that bacteria account for a great deal, but not all of it. “We had the suspicion, as have others, that the key marine ammonia oxifiers had not been discovered,” Stahl says.

Eleven years ago, when he was studying water samples from the Shedd Aquarium, he noticed that ammonia was being purged from the system. He couldn't find any sign of the usual bacteria that do this, but was unable to see what was doing the job.

Some seven years later, Stahl's group collaborated with John Waterbury at the Woods Hole Oceanographic Institute in Massachusetts to look at nitrification in the Plum Island Sound estuary. After Stahl examined the results that his postdoc, Anne Bernhard, had obtained from the field, he experienced deja vu. “It was the same observation as at Shedd,” he says. “We weren't seeing the bacteria associated with nitrification.”

Archaea became the number one suspects, but to confirm their guilt, Stahl needed to culture them and see them in action. But culturing Archaea is difficult: the microorganisms grow slowly, and bacterial by-products tend to linger for a long time, which makes getting a pure sample a challenge.

Stahl and his team decided to maximize their chances of success by taking samples from a setting where the population would be ‘enriched’ because of a high ammonia content. They went for the Seattle Aquarium. “With an aquarium full of fish poop, we thought our opportunities of isolating Archaea would be better than from a cold marine system,” Stahl says.

Bernhard set up cultures using samples from the aquarium and, after many months, was finally able to get them to grow. She also developed a method to quantify the microorganisms and determined that the production of nitrite from ammonium coincided with the growth of the Archaea. But without a pure culture, she couldn't be certain.

But then a paper appeared in Science on a genomics screen of the Sargasso Sea ecosystem. One paragraph in the article noted that a gene in Archaea was similar to one in bacteria that are known nitrifiers. “That was when we started to worry that the cat was out of the bag,” Stahl says. But rather than publish quickly, Stahl decided to continue until the Archaea culture was virtually pure.

Stahl expects to face more competition as his research continues. Other groups are looking at nitrification in soil Archaea and he expects that some are also looking at those in fresh water. Stahl says that he is keen to learn which genes are responsible for nitrification in the Archaea, and wonders if different genes are responsible in different kinds. But he hopes that finding out doesn't take as long as pinpointing the process to Archaea in the first place.