As a primary nutrient required for all living things, the forms of available nitrogen (N) present in the environment play a large role in determining where, when and how ecosystems function. Transformations among different forms of nitrogen are primarily thought to be driven by direct metabolic processes of microbes (bacteria, archaea, fungi). However, these different forms, especially intermediate forms, also exhibit a high degree of chemical reactivity. This project was designed to investigate how reactive forms of nitrogen may interact chemically with various forms of manganese (Mn) a metal commonly found in abundance in soils, sediments and natural waters. Central to this project was also the application of stable isotope analysis of nitrogen and oxygen (many nitrogen compounds are oxygen containing oxyanions, such as nitrate (NO₃^-), nitrite (NO₂^-)), as a possible tool for helping to differentiate chemical and biological reactions under natural and/or laboratory culture conditions.

During this project we conducted an initial series of various laboratory experiments to examine the reactivity of various combinations of N and Mn species, characterizing the nature of the reaction products and the dynamics of stable isotope fractionation. Mn commonly exists in one of three oxidation states (II, III, IV). Lab experiments determined that soluble forms of oxidized Mn (III), specifically those bound to an organic molecule (ligand) were very potent oxidants of intermediate forms of nitrogen, including nitrite and hydroxylamine, common products of biological activity. Interestingly, the N isotope fractionation (the degree of difference in the reaction of the heavy and light isotopes) of nitrite oxidation by Mn(III) caused a unusual inverse isotope effect, which has only previously been reported once – also for nitrite oxidation by the bacterially catalyzed reaction. The source of the additional oxygen atom was determined to originate from ambient water (not molecular oxygen) – and the reaction proceeded regardless of the presence or absence of molecular oxygen. In contrast, under biologically mediated nitrite oxygen, molecular oxygen is required for the microbial process. Thus, the oxidation of nitrite to nitrate can proceed in the absence of oxygen, where oxidized forms of Mn may be present. This finding has possible implications for better understanding how the evolution of an oxygen-bearing atmosphere on Earth, might be tied to and/or reflected Earth’s the nitrogen cycle. Similar experiments also demonstrated that a N intermediate of ammonia-oxidation (the first step of nitrification) called hydroxylamine, is highly reactive with soluble forms and mineral forms of oxidized Mn(III) and Mn(IV). In these reactions, hydroxylamine was rapidly consumed yielding both nitrite and nitrous oxide (N₂O), a potent greenhouse gas. However, experiments conducted with ammonia-oxidizing bacteria grown in the presence of Mn(III) and Mn(IV) did not appear to interfere with the purely biologically driven reaction – suggesting that the intermediate hydroxylamine does not generally have the opportunity to react outside of the microbial cells.

Looking to the nature of such coupling reactions in the environment, we also successfully culture several dozen microbes capable of oxidizing Mn and/or reducing nitrogen compounds. These cultures have been purified will continue to be characterized in future work.

The project also supported efforts to further develop quantitative numerical models of nitrogen cycling and isotopic dynamics in natural aquatic sediment water interface environments. Specifically, a numerical model that included 11 separate microbial metabolisms and/or abiotic reactions was developed for application to multi-compound, multi-isotope studies of nitrogen cycling. This model can be used to simulate how variations in all of these processes would be manifest in the natural distribution of isotopes in an environment – offering a powerful tool for examining complex coupling of a wide variety of conditions and processes. The model can also be inverted, whereby isotope data that is collected and measured can be used to provide estimates of specific rates of microbial and/or abiotic processes. To our knowledge this model represents an important advancement in our ability to quantitatively use natural isotope data towards understanding specific metabolic processes in the environment. Coupling of this approach with ‘omics-based’ approaches in the future should provide a powerful combination.

This project supported the experiential education of seven graduate and undergraduate students. These opportunities provided these students with training in cutting edge laboratory and experimental techniques in chemistry and microbiology.

Last Modified: 09/13/2021
Modified by: Scott D Wankel