Researchers at the California Institute of Technology (Caltech) have
identified an unexpected metabolic ability within a symbiotic community
of microorganisms that may help solve a lingering mystery about the
world’s nitrogen-cycling budget.
A paper about their work appears in the October 16 issue of the
The element nitrogen is a critical part of amino acids, the building
blocks of proteins, and therefore essential to all life. Although
nitrogen is plentiful on Earth—it comprises 78 percent of the
atmosphere, by volume—the element is usually found strongly bonded to
itself, in the form of the diatomic gas N2. To be
biologically useful, a nitrogen atom must be released from this coupling
and converted to a reduced, or "fixed," state; reduced nitrogen atoms
gain an electron, which makes them chemically reactive.
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
The research in the paper, "Deep-Sea Archaea Fix and Share Nitrogen
in Methane-Consuming Microbial Consortia," was supported by the National
Science Foundation and the Gordon and Betty Moore Foundation.
Source : California Institute of Technology