Explorations of Oxygen-Free Seas on the Oceanus (Part III: Nitrogen)

This is the third blog post of a series on Jennifer Glass’ experience at sea.
Read previous posts here and here

“Water, water, everywhere, Nor any drop to drink.”
- Samuel Taylor Coleridge, The Rime of the Ancient Mariner

Like water, nitrogen is everywhere, and most of it is inaccessible to life. Almost 80% of Earth’s atmosphere is nitrogen gas (N≡N), composed of two nitrogen atoms bound tightly together with a strong triple bond. But biology can’t incorporate N2 gas into organic compounds; instead, it needs nitrogen bound to three hydrogen atoms (free NH3 is ammonia; when it’s bound to organic compounds, it’s called an “amine” group).

As far as scientists can tell, no eukaryote – not a single plant, animal, or fungi on the face of the Earth – can break the N≡N triple bond enzymatically. Only a subset of single-celled Bacteria and Archaea contain the special enzyme that does the complex and energetically expensive chemistry to convert N2 gas to biologically available ammonia (NH3), making these microbes of vital importance to agriculture and global elemental cycles. 

On the other end of the nitrogen cycle, nitrogen is returned to its gaseous form. This process, called “denitrification,” proceeds by a series of steps, catalyzed almost solely by bacteria. While we humans can only respire oxygen, anaerobic bacteria are far more creative chemists. Many OMZ bacteria “inhale” oxidized nitrogen and “exhale” more reduced gaseous forms (NO, N2O and N2). Although OMZs contain only one one-thousandth of the total ocean volume, one third of the ocean’s “fixed” nitrogen is lost back to gaseous N2 in these anoxic water columns, making them central players in the global nitrogen cycle. 

On this cruise, PhD student Philipp Hach, from Professor Marcel Kuypers’ group at Max Plank Institute (MPI) for Marine Microbiology in Bremen, Germany, measured the rates at which fixed inorganic nitrogen is lost from OMZ waters by tracking the rare stable isotope 15N into N2 gas. He added alternative organic nitrogen compounds containing an amine group in addition to inorganic nitrogen substrates. Back at MPI-Bremen, he will measure 15N2 gas and calculate the rate of nitrogen loss from OMZ waters. 

Chief Scientist Frank Stewart and his Georgia Tech marine microbiology research group study the microbes that making a living off of nitrogen conversions in OMZs. Last year, they discovered that Pelagibacter (the same bacterial genus we covered in my first post) are responsible for the first step in nitrogen loss in OMZs, the reduction of nitrate (NO3-) to nitrite (NO2-; Tsementzi et al., Nature, 2016). Cory Padilla, a PhD candidate in the Stewart lab and cruise participant, recently found numerous active genes from NC10 bacteria in OMZs (Padilla et al., 2016, ISME J). These microbes use nitric oxide (NO) to make their own oxygen in anoxic ecosystems. Postdoc and cruise scientist Dr. Anthony Bertagnolli is using genomic tools to study new uncultivated microbial phyla that produce the NO2- that feeds NC10 bacteria.

At every station, Professor Bo Thamdrup of the University of Southern Denmark and Masters student Herdis Gudlaug Steinsdottir of the Technical University of Denmark collect samples for nutrient depth profiles. To measure NO2-, they combine the seawater samples with chemicals under acidic conditions to form a pink azo compound to quantify the NO2- down to nanomolar concentrations.

This “Griess” assay was discovered almost 150 years ago (Griess, 1879), and was recently optimized for small volumes by cruise participant Dr. Emilio Garcia-Robledo of Aarhus University in Denmark and Cadiz University in Spain (Garcia-Robledo et al., 2014).

A predictable spike in NO2-, visible in the Griess test as the brightest pink sample, is found at every station towards the top of the OMZ, a product of anaerobic microbes breathing NO3-. Now Garcia-Robledo is researching the source of O2 used by aerobic microbes at the top of the OMZ (Garcia-Robledo et al., 2017).

My own lab, the Glass lab at Georgia Tech, is focused on interactions between biotic and abiotic processes in greenhouse gas cycling. Nitrous oxide (N2O), also known as “laughing gas” is a potent greenhouse gas and a key intermediate in the nitrogen cycle. Under fully anoxic conditions, bacteria can convert nitrous oxide to N2 gas. But when even traces of O2 are present, such as at the oxic-anoxic interface above the OMZ, N2O accumulates. We hypothesize this N2O is produced by a combination of biological and chemical processes at this interface (see recent paper: Zhu-Barker et al, 2015). 

To study this process, Georgia Tech Ocean Science & Engineering PhD student Abbie Johnson and I sampled every five meters through the oxic-anoxic gradient, also known as the “oxycline”, at every station. We preserved these samples with mercuric chloride to stop all biological activity, and will take them back to our lab in Atlanta to measure N2O and another highly reactive player in the nitrogen cycle, hydroxylamine, which my PhD student Amanda Cavazos is studying as a potential source of this greenhouse gas. We are also collaborating with PhD student Katharina Kitzinger and Dr. Laura Bristow at the Max Plank Institute for Marine Microbiology for cell counts of nitrogen-cycling microbes that may be involved in producing reactive intermediates that may ultimately end up as laughing gas. 

Stay tuned for the final blog post from our expedition featuring scientists from the expedition and the exciting research that inspires them to go to sea.

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