The regulation of bacterial nitrous oxide reduction

Humans are entirely dependent on the oxygen we breathe to support our life 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, rather than oxygen to support life. One of these life-supporting processes is denitrification, in which water-soluble nitrate is converted to gaseous products, nitric oxide, nitrous oxide (N2O) and dinitrogen. This denitrification process can take place extensively in agricultural soils where nitrogen rich fertilisers added to stimulate plant growth can also stimulate bacterial life. Soil bacteria that can denitrify need to protect themselves from the effects of NO, a potent toxin, produced through their own metabolism. They have an enzyme called 'nitric oxide reductase' that has evolved to keep NO levels low in the bacteria by converting it to the relatively benign nitrous oxide (N2O) which can sometimes be released into the atmosphere. This bacterial survival strategy has significant environmental consequences as N2O is a potent greenhouse gas which can damage the ozone layer.

When discussing greenhouse gas emissions the general 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 be a cause for public and political 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 20% and this atmospheric loading is increasing further by 0.25% each year. 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 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 understood 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 depend on metals such as molybdenum, iron and copper for their activity. The enzyme that breaks down N2O to inert N2 is a copper-containing enzyme called nitrous oxide reductase. It is the major enzyme on the planet that is responsible for the destruction of the potent N2O greenhouse gas. Without it the atmospheric levels of N2O would be much greater that they currently are. The nitrous oxide reductase contains twelve atoms of copper and so its activity in the environment is highly 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.

As a result of the application of nitrogenous fertilisers, agricultural soils are the largest source of anthropogenic N2O. 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 fundamental studies on model organisms in controlled laboratory environments. This programme represents just such a study focused on the mechanism by which copper regulates N2O emission.

Nitrous oxide (N2O) has a ~300-fold greater global warming potential than carbon dioxide and has been described as the biggest single cause of ozone depletion over the arctic. Its atmospheric loading is increasing by ~0.25% each year, and it has a very long atmospheric lifetime of ~150 years. N2O is estimated to contribute up to 9% of the global radiative forcing of greenhouse gas emissions. Agriculture accounts for ~70% of anthropogenic atmospheric loading of N2O. Understanding the environmental factors that control N2O production and consumption by microbes is a critical and major challenge on the road to developing practical mitigation strategies for N2O emissions.

Denitrifying bacteria play an important global role in the synthesis and consumption of N2O, but very little work has been done on studying the regulatory networks that modulate the assembly and activity of the biochemical apparatus that catalyses production and destruction of this greenhouse gas. As arable lands become more intensively exploited Cu-deficiency is also becoming a more acute global concern. Critically, N2O reduction is dependent on the Cu-containing enzyme nitrous oxide reductase (NosZ).

We have shown that under nitrate-rich, Cu-depleted conditions denitrifying bacterial cultures release N2O at >100 times the rate of Cu-replete cultures. We have undertaken the first global transcription analysis of a denitrifying respiratory network and this has shown a clear regulation of the nos genes by Cu, which leads to the down-regulation of the NosZ enzyme. The resulting impact on N2O release is very pronounced with 40% of nitrate input into the system being released as this potent greenhouse gas. It is therefore important study the regulatory network of N2O emission to resolve: (1) the mechanism by which Cu controls N2O reduction and (2) how regulation by Cu integrates into regulation by other key environmental variables such as oxygen and the Carbon/Nitrate ratio.

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, in particular the response to availability of copper. The impact of this work will be the establishment of the link between copper and the expression of proteins needed for a functional nitrous oxide reductase. There will be diverse beneficiaries of knowledge arising from this research including 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 addition there will be academic beneficiaries in subject areas ranging from Environmental Science to Biochemistry. 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 of this release, particularly the step of N2O reduction, either at the level of the gene or the N2O reductase protein, yet this enzyme is central to any strategy for the mitigation of N2O emission. A low activity will lead to higher emissions. 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 nitrous oxide reduction 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. Professor Richardson coordinates a nitrous oxide focus group that has received press coverage world-wide. This focus group will be a major conduit for dissemination of the outcome of this research. We will ensure that major developments are publicised on the regularly updated University web sites. The research will be published in high impact journals and oral communications given at international conferences. All investigators will take every opportunity that arises to talk to the general public about nitrous oxide emissions and the underlying biology.