Every year, the Mississippi River dumps around 1.4 million metric tons of nitrogen into the Gulf of Mexico, much of it runoff from agricultural fertilizer. This nitrogen can lead to algal blooms, which in turn deplete oxygen concentrations in the water, creating hypoxic dead zones. The nitrogen cycle is a phenomenon environmental scientists would really like to understand better.
“As humans, we do put a lot of reactive nitrogen compounds into the ocean, especially in coastal regions, by…river runoff,” said Katharina Kitzinger of the Max Planck Institute for Marine Microbiology in Bremen, Germany. “It’s really crucial to understand how microbes turn over this excess nitrogen that we put into the environment.”
Nitrification is a two-step part of the nitrogen cycle in which ammonia is converted into nitrate. Nitrification has been understood since the late 19th century to be conducted by an array of microbes that first oxidize ammonium into nitrite and then nitrite into nitrate.
The details of this process have remained somewhat opaque until recently. It was only in 2005 that some of the key organisms in the first step of nitrification, ammonia-oxidizing archaea, were discovered. These archaea, belonging to the phylum Crenarchaeota, are among the most populous cell types in the oceans, and their ability to oxidize ammonia filled in one of the major blank spots in scientists’ understanding of the nitrogen cycle.
But this led to another mystery: Ammonia-oxidizing archaea are quite numerous, composing up to 40% of the oceanic microbe community, but the nitrite-oxidizing bacteria crucial to the second step in nitrification are 10 times less abundant.
“There’s hardly any nitrite in the oceans, which really suggests that any ammonia, which is oxidized to nitrite, must be balanced by the second group of organisms, which are converting the nitrite to nitrate,” Kitzinger said. “That was basically the huge discrepancy. We discovered these hugely abundant ammonia oxidizers, but the known nitrite oxidizers typically just make up” a small percentage of oceanic microbial life.
There were hypotheses to explain this discrepancy, according to Hannah Marchant, Kitzinger’s colleague in Bremen. Oxidizing nitrite yields less energy than oxidizing ammonium, so it’s possible that nitrite-oxidizing bacteria are simply slower growing. There could also be an undiscovered nitrite-oxidizing organism as abundant as the ammonia-oxidizing archaea, which would not be unprecedented given not only the recent discovery of the archaea themselves but also the 2015 discovery of new terrestrial nitrogen oxidizers, the comammox organisms.
Efficient, Fast-Growing Bacteria
But a new paper published in February in Nature Communications, of which Kitzinger and Marchant are first and second authors, respectively, suggests that there are no new nitrite oxidizers to be found in the oceans. Instead, existing nitrite-oxidizing bacteria are the key players and are unexpectedly vigorous ones at that.
Ever wondered why marine #AOA are much more abundant than #Nitrospinae?
Check out our new paper in @NatureComms https://t.co/OaIgt430FK@BiogeoMPIBremen, @DOME_Vienna, SDU, Georgia Tech pic.twitter.com/Cu7XwHzrMU
— Kathi Kitzinger (@KathiKitzinger) February 7, 2020
Nitrite-oxidizing bacteria, they write, “are more energy efficient, and grow faster than [ammonia-oxidizing archaea],” at least within the Gulf of Mexico where the researchers based their study. “If our results can be extended to the rest of the ocean, no additional undiscovered [nitrite-oxidizing bacteria] are required to account for the global oceanic balance between ammonia and nitrite oxidation.”
The finding came as something of a surprise to Kitzinger and Marchant and their colleagues at the University of Vienna, the University of Southern Denmark, and the Georgia Institute of Technology. They had originally set out to the Gulf of Mexico to study archaea’s utilization of nitrogen compounds, particularly urea and cyanate. “But we also looked at the utilization of these compounds in nitrite oxidizers,” Kitzinger said, focusing on nitrite-oxidizing bacteria in the phylum Nitrospinae. Using isotopically labeled urea and cyanate to measure uptake by sampled microbes, as well as cell size and number, researchers were able to calculate cell growth rates.
“What we then saw was that the nitrite oxidizers actually incorporated a lot more of these compounds than the ammonia oxidizers” and were more energy efficient and faster growing than the archaea, Kitzinger said. In the most active water column samples, the bacteria increased fivefold over 24 hours.
A New Set of Bugs
But those findings did raise some questions for other researchers in the field.
Bess Ward is the William J. Sinclair Professor of Geosciences at the Princeton Environmental Institute in New Jersey and has been studying nitrogen her entire career. “They say that during a 24-hour incubation, some of the abundance is increased by five[fold] or sixfold, and that means a specific growth rate of several per day,” Ward said. The hypothesis struck her as strange because the measured growth rates of related Nitrospinae are significantly slower. “That actually stood out as a ‘Wow, so if that’s true, then they’ve got a new set of bugs.’”
The measured growth rates also stood out to Maria Pachiadaki, a researcher at the Woods Hole Oceanographic Institution in Massachusetts whose work focuses on carbon fixation in the dark ocean. “Within 24 hours, it seems a bit unusual for a specific group to, you know, to increase like five[fold] or sixfold in abundance,” she said. It’s not something Pachiadaki believes casts doubts on Kitzinger and Marchant’s results overall, “but it’s something to keep in mind and try to see whether these incubations are outliers and need to be removed from the downstream analysis.”
But Kitzinger pointed out that her team measured growth rates two different ways—by counting cells in samples and by isotope uptake—and those rates matched, so “we don’t think there is a measurement artifact.” She also noted that although her team did see a maximum fivefold increase over 24 hours, the average across all their samples was a less extravagant 1.7.
“One also has to keep in mind that the shelf of the Gulf of Mexico is characterized by exceptionally high nutrient fluxes and ammonia and nitrite oxidation rates,” Kitzinger said. “We would expect that in the open ocean, where nitrification activity is much lower, Nitrospinae will also grow accordingly slower.”
Other unique properties of the Gulf could also be factors; Ward suggested that the relatively warm waters could be responsible for “these extravagant growth rates.”
Another question raised by Kitzinger and Marchant and their colleagues’ work awaits further research to resolve: If the nitrite-oxidizing bacteria are so efficient and vigorous, why are they so much less numerous in the oceans? The obvious answer, they said, would be high mortality and population turnover, but mortality due to what is not yet clear.
“At the moment, we don’t know whether that mortality is through grazing, that there’s something that’s just really eating them a lot, or whether they have a very high rate of viral infection,” Marchant said. “That is something that you would have to go back and do specifically designed studies to look at.”
That answer could help researchers better appreciate the broader implications of this new understanding of nitrite-oxidizing bacteria, according to Pachiadaki. Her own research has focused on how bacteria and other microbes fix carbon in the dark ocean, where there are no photosynthesizing organisms. That work led Pachiadaki to realize she was missing a large group of organisms that must be contributing to dark carbon fixation and, eventually, to the conclusion that the missing organisms must be the nitrite oxidizers.
“If we can also verify that this carbon is also highly and actively grazed, then we also have the link of how this carbon, that is produced chemoautotrophically, moves to a higher trophic level,” she said. “[We can understand] how carbon is transferred and how carbon can sustain a food web there.”
“The oceans are still such an unknown in a lot of ways that we don’t really know their contribution to carbon fixation,” Marchant added. “If we don’t understand the basic fundamentals of how these microorganisms function, then it’s also very hard to predict what might happen in the future.”
That’s a thread that Pachiadaki, for one, is ready to pull. “I have actually emailed [Kitzinger] in order for us to get the carbon succession rate per cell,” she said. “I want to see how comparable it is with what I calculated. So this is an ongoing discussion.”
—Jon Kelvey (@JonKelvey), Science Writer