The development of a method using denitrifying bacteria Ochrobactrum anthropi for δ15N analysis of nitrate

Abstract

Nitrogen (N) is a major element in protein synthesis and is often the limiting nutrient in aquatic environments. As N availability is tightly coupled with carbon (C) uptake, it also has the potential to impact on species diversity, ecosystems and climate should levels be altered. Fuel combustion and synthetic fertilizers are now increasing N levels and perturbing N cycles, highlighting the need to manage anthropogenic N in order to reduce any negative effects. Tracing N levels back to source point can be achieved by the study of stable N isotopes that pool in unique 15N/14N ratios during biogeochemical processes. Isotope ratio analysis is considered a powerful tool but can involve complicated chemical procedures resulting in fractionation and erroneous results when sample nitrate (NO3-) and nitrite (NO2-) is extracted for δ15N measurements. Such problems may now be avoided by recently developed bacterial methods that manipulate bacteria into denitrifying NO3- and NO2- to nitrous oxide (N2O) which can be analysed directly on a mass spectrometer. The current project trialled two bacterial methods for denitrification of three NO3- standards with known 15N/14N ratios using the bacterium Corynebacterium nephridii which produced unsatisfactory results. This was attributed to culture conditions affecting denitrification rates, as the methods had been developed using two denitrifying Pseudomonas species, P.aureofaciens and P.chlororaphis. A further complication arose when the candidate bacterium C.nephridii was found to be a totally different species of denitrifier, Ochrobactrum nephridii, but work continued using this species as the bacterium could potentially denitrify N-oxides from polluted and saline samples. This resulted in the development of a new method that produced 15N/14N values as well as 18O/16O values which were in good agreement with known values of standards, demonstrating that the method is suitable for isotope analysis of δ15N as well as δ18O in freshwater samples.