Ettwig et al. 2009

Ettwig KF, van Alen T, van de Pas-Schoonen KT, Jetten MSM, Strous M. 2009. Enrichment and molecular detection of denitrifying methanotrophic bacteria of the NC10 phylum. Applied and Environmental Microbiology 75: 3656-3662.

These authors describe a series of experiments and procedures designed to investigate an enigmatic organism known as NC10, a bacteria in its own eponymous phylum that currently represents the only demonstrated case of biological reduction of nitrate coupled to oxidation of methane under anaerobic conditions. While anaerobic methane consumption has been observed in some archaea, it has not been found coupled to denitrification.

A laboratory culture eventually dominated by NC10 organisms of group a (a distinction within the phylum) was established, based on sediment collected from a eutrophic ditch draining agricultural land on the floodplain of the Rhine river in the Netherlands. This culture was grown and maintained under conditions in which the only carbon source was the sparge gas of CH4-CO2, and nitrogen was supplied with the mineral inputs as nitrate and nitrite, along with a wide range of other inorganic compounds and trace elements.

The major finding of this study was a wealth of knowledge of the basic characteristics of the NC10 organism, and confirmation that it does indeed oxidize methane under anaerobic conditions coupled to denitrification. This process is energetically favourable, and the theoretical stoichiometry matches the observed changes in chemical composition in these experiments, with the nitrite reduction to methane consumption ratio of 8:3.5, versus 8:3 based on mass balance calculations.

One of the surprising aspects of this organism is that its methane-oxidizing activity is completely inhibited by black butyl rubber, as is found in black rubber stoppers for serum vials and other glassware. Grey or red butyl rubber stoppers do not show such inhibition, and repeated boiling of black butyl rubber stoppers in HCl did not remove the inhibitory effects. Strictly anoxic conditions are not required for all aspects of working with this organism; brief exposure to atmospheric oxygen during liquid transfer, for example, did not inhibit methanotrophic activity.

Another strange feature of NC10 concerns its 16s rDNA sequences. General 16s primers do not amplify NC10 DNA. These authors developed new primers for the 16s region based on the DNA in their culture, which they were able to confirm as NC10 based on FISH observations. The new primers allowed them to work more easily with the NC10 DNA, which is not surprising, but the sequences of NC10 16s found did not differ in critical ways from the target regions of the general 16s primers. So, it is unknown why the general 16s primers do not work on NC10 DNA.
This paper was recommended to me and I would not likely have discovered it without this recommendation. I have results from the 2009 work at Alexandra Fjord that suggest simultaneous consumption of CH4 (oxidation) and N2O (reduction), and I did not know if these two processes might be linked in a single organism or within a system such as a symbiosis or food-chain as 2 halves of a redox couple.

My results may be more suggestive of an alternate situation. Rather than anaerobic oxidation of methane (weirdness) coupled to nitrate reduction, I may be looking for cases of reduction of nitrous oxide under aerobic conditions (weirdness) coupled to methane oxidation.