Understanding nitrous oxide emission from denitrifying bacteria: integrating chemostat and soil studies

We humans we are entirely dependent on the oxygen we breath to support our metabolic processes. Significantly, this is not so for many species of bacteria. Faced with a shortage of oxygen in their environment many bacterial species are able to switch to using nitrate (NO3-), rather than oxygen to support respiration. One of these energy yielding processes, known as denitrification, converts water-soluble nitrates to gaseous products, nitric oxide (NO), nitrous oxide (N2O) and dinitrogen (N2). This denitrification process can take place extensively in agricultural soils where nitrogen rich fertilisers added to stimulate plant growth can also stimulate bacterial nitrogen cycling. Soil bacteria which can denitrify need to protect themselves from the autotoxic effects of NO produced through their own metabolism. They have an enzyme, nitric oxide reductase (NOR) that has evolved to keep endogenous NO levels low by converting it to the relatively benign nitrous oxide (N2O) which can be released into the atmosphere. From the perspective of bacterial metabolism the job of detoxifying the cytoxic NO is done when it is converted to N2O, but from an environmental perspective an envirotoxin, a greenhouse gas, has been produced. When discussing greenhouse gas emissions the public are acutely aware of the problems posed by carbon dioxide and methane. However, emissions of N2O, perhaps best known as the dental anaesthetic 'laughing gas', should also cause concern. N2O was first discovered by the British chemist Joseph Priestley in 1793 when its atmospheric levels had been steady for millennia. However, over the last 100 years N2O in the atmosphere has increased by 50 parts per billion and this atmospheric loading is increasing further by 0.25% each year, with most commentators linking this increase to intensive use of fertiliser to increase farmland productivity in the 20th Century. Although its atmospheric levels are only a fraction of that of CO2 it has a 300-fold greater global warming potential. Thus when expressed in terms of CO2 equivalents it represents around 10% of total global emissions of greenhouse gases. Since it has an atmospheric lifetime of some 150 years the N2O produced today will potentially influence the climate experienced by our great-great grandchildren thus it is important to devise strategies to mitigate these releases now. The pathways by which denitrifying bacteria produce NO from nitrate are undertstood from a molecular level with structures of enzymes that convert nitrate to nitrite (nitrate reductases) and nitrite to nitric oxide (nitrite reductases) being known. These enzymes are metalloproteins that depend on transition metals such as molybdenum, iron and copper for activity. The enzyme that breaks down N2O to inert N2 is a copper-containing enzyme (Nos). It is the major enzyme on the planet that is responsible for the potent N2O greenhouse gas. Without it the atmospheric levels of N2O would be much greater that they currently are. The molecular structure of Nos is known. It contains twelve atoms of copper and so its activity in the environment is dependent on the bioavailability of copper. It is also sensitive to pH and oxygen and so its activity in the environment is dependent on a number of different environmental variables. The largest source of anthropogenic N2O emissions is agricultural soils because of the application of nitrogenous fertilisers to soils. Since the UK signed up to the Kyoto Protocol, many non-biological sources of N2O emissions have been reduced, but emissions from biological sources are less easy to manage. Efforts to improve the prediction and management of agricultural N2O emissions will benefit from a better understanding of the factors that influence the net production of N2O by bacteria. This requires a combination of studies on model organisms in controlled laboratory environments and on studies in situ in soils. This project will provide such an a integrated study.

Nitrous oxide (N2O) is a potent greenhouse gas, and based on the IPCC approved unit of CO2 equivalent is estimated to contribute around 10% to global greenhouse gas emissions. Consequently it is recognized in the Kyoto Protocol that it is essential to mitigate these releases. Agricultural soils are the main source of atmospheric N2O, with the microbial process of denitrification usually being predominant in this production. Denitrification is the only microbial pathway with the proven ability to further reduce N2O to the environmentally harmless N2. This holds an essential, but as yet unexplored, key to mitigation of N2O, in which we enhance the microbiological process, rather than lowering or eliminating its activity or denitrifier diversity. The pathways by which denitrifying bacteria produce nitric oxide from nitrate are now well understood at a molecular level with molecular structures of enzymes that convert nitrate to nitrite (nitrate reductases) and nitrite to nitric oxide (nitrite reductases) being known. However, there is actually very little understanding of the regulation of the only enzyme that makes a significant global contribution to the consumption of nitrous oxide, the bacterial nitrous oxide reductase. Understanding the regulation of this enzyme will enable the development of soil management strategies for N2O mitigation. Here, Analysis of N2O production under varying pH, O2, mineral and C availabilities in model denitrifying bacteria will be combined with 15N-enrichment techniques to quantify N2O-to-N2 ratios, and molecular techniques to relate these emissions to expression of the Cu-NO2 and N2O reductases in situ in soil under different soil organic matter and pH management, and Cu-sulphate additions, enabling us to identify, for the first time, the optimum conditions for maximum reduction of N2O to N2.

The research programme focuses on the regulation of the release, by bacteria, of a potent greenhouse gas. Increases in the concentrations of greenhouse gases, carbon dioxide (CO2), methane (CH4), nitrous oxide (N2O), and halocarbons in the atmosphere due to human activities are associated with global climate change. In this research programme we focus on N2O. The concentration of N2O in the atmosphere has increased by 20% since it was discovered in 1793. Although atmospheric concentration of N2O is much smaller (314 ppb in 1998) than of CO2 (365 ppm), its global warming potential (cumulative radiative forcing) is 296 times that of the latter in a 100-year time horizon. Currently, it contributes about 10% of the overall global warming effect. Of that, almost 80% of N2O is emitted from agricultural lands, originating from N fertilisers (32%), soil disturbance (38%), and animal waste (30%). Nitrous oxide is primarily produced in soil by the activities of microorganisms during the denitrification process that converts nitrate to N2O and N2 gas. The ratio of N2O to N2 production depends on oxygen supply or water-filled pore space, decomposable organic carbon, N substrate supply, temperature, and pH. However, the cellular metabolic level at which these factors regulate the N2O:N2 ratio is not understood. The impact of this work will be to use two model denitriying organisms to increase our understanding of how the enviroment regulates the N2O:N2 ratio at the molecular and cellular level in controlled laboratory systems and assess to what extent this translates into observation made of denirification activities in soil cores. The main beneficiaries of knowledge arising from this research will be organisations such as the Environment Agency (EA), Defra, Natural England, The Arable Group and Local Authorities (LA) and other bodies (including the farming community) with an interest in the management of the impact of agriculture on nitrous oxide release. In the context of LA's, there will be increasing pressure for regions to demonstrate their contribution to greenhouse gas mitigation strategies and carbon reduction compliance, possibly in future through carbon taxation/credit schemes. Globally we stand on the brink of some major opportunities in agriculture and food production for lowering the production of greenhouse gases, such as N2O. For example, the current interest in production of crops for second generation biofuels brings with it a need to understand the environmental controls and consequences of intensive crop production. Little is known of the direct and indirect effects on greenhouse gases of bioenergy crop and biofuel production, but estimates are that N2O is the largest greenhouse gas source in these systems. Surprisingly little is known about this regulation, particularly of N2O reduction, either at the level of the gene or the protein of N2O reductase, yet this enzyme is central to any strategy for the mitigation of N2O emission. Moreover since denitrification is now being shown to be more widespread in its occurrence than previously thought, it is unlikely that it will ever be possible to develop farming practices that completely eliminate denitrification from agriculture. We propose that a more reliable approach to mitigating N2O emissions is to translate our emerging knowledge of the enzymology of denitrification into protocols designed to manipulate the physiology of denitrifying bacteria so that the extent of reduction of N2O to N2 is not frustrated but increased.