Caltech researchers reveal unexpected sources of nitrogen fixation

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Although lightning, combustion, and other nonbiological processes can reduce nitrogen, far more is generated by nitrogen-fixing microorganisms such as bacteria-in particular, photosynthetic cyanobacteria. These organisms produce the bulk of the nitrogen available to living things in the ocean.

Still, when researchers add up all of the known sources of fixed nitrogen (biological and otherwise) in the global nitrogen cycle and compare it to the sinks-where nitrogen is taken up for growth and energy-they come up short. It appears that more nitrogen is being used than is being made. The apparent nitrogen budget, in effect, does not balance. This discrepancy had led scientists to question whether the nitrogen cycle is truly out of balance, or whether the known inventories of sources and sinks are misleadingly incomplete.

Victoria J. Orphan, an assistant professor of geobiology at Caltech, along with graduate student Anne E. Dekas and postdoctoral research scholar Rachel S. Poretsky, suggest the answer is, at least in part, an incomplete catalog of the sources of fixed nitrogen.

The team studied ocean sediment samples obtained in methane cold seeps located at a depth of about 1,800 feet. The area, known as the Eel River Basin, is located approximately 20 miles off the coast of the northern California town of Eureka, on a continental margin in a region supporting high levels of natural methane seepage at the seabed.

In the laboratory, the researchers examined the methane-rich sediment and the tiny microbial conglomerations that live within. These spherical cell conglomerates, averaging about 500 cells each, consist of two types of anaerobic microorganisms living in a unique symbiotic relationship fueled by methane. The first microorganism is a bacterium that reduces the chemical sulfate into sulfide (via a process that produces the rotten-egg odor of salt marshes and mud flats) to generate energy. The second is a methane-oxidizing archaeon (the archaea are a group of nonbacterial single-celled microorganisms). Working together, these two symbionts are responsible for consuming the majority of the naturally released methane in the deep sea.

Although these symbiotic associations themselves are not new-these conglomerations were discovered about a decade ago and are found on continental margins worldwide-the Caltech scientists discovered something unexpected: the methane-consuming archaea were actively fixing nitrogen, and sharing it with their bacterial neighbors.

"This is the first time that nitrogen fixation has been documented within methane-oxidizing archaea," Dekas says.

Interestingly, although these organisms have a nitrogen-poor diet of methane gas, they live in an environment that contains reduced nitrogen-in the form of ammonium and other chemicals-which means they shouldn't need to create their own. "It's possible that they do need to because they are living in a crowded community-a tightly packed ball-that prevents some organisms from having access to the nitrogen," she says. Another possibility is that these environments do not have as much biologically available reduced nitrogen as had been thought.

To determine that the archaea were indeed fixing nitrogen, the researchers first incubated the archaeal-bacterial assemblages with a dinitrogen gas, N2, that was composed of two atoms of nitrogen-15. Nitrogen-15 is a nonradioactive isotope of nitrogen that contains one more neutron than regular nitrogen (nitrogen-14) and can be used as a tracer for the incorporation of the element.

The researchers then used a technique called fluorescent in situ hybridization (FISH) to stain the two types of organisms in the sediment, and analyzed these cells for their nitrogen-15 content using a state-of-the-art instrument called a nanometer secondary ion mass spectrometer, or nanoSIMS. The nanoSIMS, which is housed at the Caltech Center for Microanalysis, is capable of collecting chemical and isotopic data at a spatial scale of 50 to 100 nanometers, or around five to 10 times smaller than the size of a single microbial cell.

Both the archaea and, to a lesser extent, their bacterial neighbors had incorporated the nitrogen-15, which could have happened only if the N2 had been fixed by the archaea-and then shared.

"The high spatial resolution of the nanoSIMS instrument-which produces a focused beam of ions that is smaller than a single cell-allowed us to directly pinpoint which of the symbiotic cells in the consortia had assimilated the nitrogen-15-labeled N2 into their biomass," Orphan notes.

The fixation process, say the scientists, is painfully slow; the organisms themselves have ultra-slow growth rates, doubling once every three to six months. "But they are passing on some nitrogen to their neighbors, which means they are producing more than they need," despite the energy cost of doing so, Dekas says. "We don't know what benefit the archaeal organisms get from sharing it, but we do know they need the bacterial symbiont to stay alive," she adds.

"Previously, assumptions about when and where nitrogen fixation takes place made it seem unlikely that nitrogen fixation would occur in this environment, or within such energetically starved organisms," Dekas says. "These results suggest that these assumptions may need to be reevaluated, and that there could be more nitrogen-fixing organisms in other unexpected environments. Together, these previously overlooked sources of nitrogen may be an important component in the marine nitrogen inventory."

California Institute of Technology