Biochemical Society Transactions

Biochemical Society Focused Meeting

Enzymology and ecology of the nitrogen cycle

Rosa María Martínez-Espinosa , Jeffrey A. Cole , David J. Richardson , Nicholas J. Watmough

Abstract

The nitrogen cycle describes the processes through which nitrogen is converted between its various chemical forms. These transformations involve both biological and abiotic redox processes. The principal processes involved in the nitrogen cycle are nitrogen fixation, nitrification, nitrate assimilation, respiratory reduction of nitrate to ammonia, anaerobic ammonia oxidation (anammox) and denitrification. All of these are carried out by micro-organisms, including bacteria, archaea and some specialized fungi. In the present article, we provide a brief introduction to both the biochemical and ecological aspects of these processes and consider how human activity over the last 100 years has changed the historic balance of the global nitrogen cycle.

  • ammonium
  • nitrate
  • nitric oxide
  • nitrite
  • nitrogen cycle
  • nitrous oxide

Role of nitrogen in the biosphere

Nitrogen is present in the environment in oxidation states ranging from +5 to −3 as a wide variety of chemical compounds, including organic nitrogen, ammonium (NH4+), hydroxylamine (NH2OH), nitrate (NO3), nitrite (NO2), nitric oxide (NO), nitrous oxide (N2O) and dinitrogen gas (N2) [1]. The growth of all organisms depends on the availability of reduced nitrogen which is required in large amounts as an essential component of proteins and nucleic acids. Consequently, the lack of biologically available nitrogen is often the limiting factor for growth and biomass production, even in environments where there is a suitable climate and adequate water to support life.

Although the Earth's atmosphere (approx. 78% N2) is an abundant source of nitrogen, it cannot be used by most organisms because the activation energy of the triple bond between the two nitrogen atoms renders the dinitrogen molecule effectively inert. This problem is overcome by micro-organisms that have a central role in nitrogen fixation and almost all other aspects of nitrogen availability and thus support life on Earth. These interconversions of nitrogen compounds in the environment constitute the global nitrogen cycle, which maintains relatively small amounts of fixed or combined nitrogen in continuous exchange with atmospheric dinitrogen.

Nitrogen availability affects the rate of important ecosystem processes such as primary production and decomposition. In recent decades, human activities, including the use of artificial nitrogen fertilizers, fossil fuel combustion, and release of nitrogen in wastewater, have dramatically altered the balance of the global nitrogen cycle [2,3]. Consequently, researchers in a number of disciplines, including microbiologists, biochemists, soil scientists, ecologists and atmospheric chemists, working on different aspects of the nitrogen cycle, have increasingly come together to explore some of the great challenges facing 21st Century humankind, including climate change [4,5], food security [6], waste-water treatment [7] and human health [8,9].

The nitrogen cycle

The nitrogen cycle represents one of the most important nutrient cycles found in terrestrial ecosystems, and it is one of the most difficult cycles to study because there are so many important forms of nitrogen, and because many organisms are involved in their interconversion [10]. Because of the huge agricultural and environmental implications of the nitrogen cycle, both prokaryotic and plant nitrogen metabolism have become major areas of research over the last few years.

The global biochemical nitrogen cycle includes redox reactions used for assimilatory purposes or in respiratory pathways for energy conservation. The assimilatory reactions include nitrogen fixation (N2 conversion into ammonia by free-living or symbiotic bacteria) [11] and assimilatory nitrate reduction (the reduction of nitrate to ammonium) [12,13]. Both pathways produce ammonium which is incorporated into carbon skeletons for growth. Apart from the respiratory reduction of nitrate to ammonia that is widespread among anaerobic fermentative bacteria, there are two major energy-generating processes: nitrification (soil-living bacteria and other nitrifying bacteria oxidize ammonia to nitrate) [14], and denitrification (anaerobic or facultative anaerobic microbes reduce nitrate to N2 or other nitrogen gases under anaerobic conditions) [15]. To complete the global nitrogen cycle, two other pathways are carried out only by micro-organisms: ammonification (many bacteria and fungi degrade organic matter, releasing fixed nitrogen for reuse by other organisms), and anammox (anaerobic ammonium oxidation that produces N2 by reducing nitrite and oxidizing ammonium) [16,17].

As a result of this biogeochemical cycle, the percentage of nitrogen in the atmosphere remains constant: nitrogen is continually released into the air by the action of denitrifying bacteria and continually returned to the cycle through the action of nitrogen fixing micro-organisms, lightning and industrial production of artificial fertilizer.

Enzymology of the nitrogen cycle

The simplest scheme to describe a complete nitrogen cycle involves five reduction and three oxidation reactions [18]. In reality, one ecosystem or even a single micro-organism that can, for example, reduce nitrate under both aerobic and anaerobic conditions is rather complicated. Many enzyme-catalysed reactions are involved, all of which are highly regulated. Understanding the structure and mechanism of the metal-containing enzymes that catalyse the redox reactions of the nitrogen cycle [19,20] has been a major research topic over the last two decades. Considerable progress has been made with some enzymes, such as nitrite reductases [21,22], but significant challenges remain if we are to fully understand bacterial NO reduction [23] and the assembly of the N2O reductase [24], while the enzymology of anammox remains poorly described.

Progress in understanding the biochemistry of the nitrogen cycle has been complemented by the availability of the complete genome sequences of hundreds of micro-organisms. This has enabled new approaches to understanding their comparative physiology and ways in which individual species regulate nitrogen metabolism [25,26]. Comparison of the derived amino acid sequences of genes encoding nitrogen-cycle proteins has allowed the evolution of this important cycle to be analysed across the three domains of life [27,28]. More recently, metagenomic sequencing of an enrichment culture that couples the anaerobic oxidation of methane with the reduction of nitrite to dinitrogen has pointed to the existence of previously undescribed enzyme activities [29].

Ecology of the nitrogen cycle

The store of nitrogen found in the atmosphere is about one million times larger than the total nitrogen contained in living organisms. Other major stores of nitrogen include organic matter in soil and the oceans. Almost all of the nitrogen found in any terrestrial ecosystem originally came from the atmosphere. Although significant amounts enter the soil in rainfall or through the effects of lightning [30], the majority of nitrogen is biochemically fixed within the soil by specialized micro-organisms that live symbiotically with plants. The bacteria live in a specialized microenvironment provided by the plant (root nodules) in which they can reduce nitrogen, some of which is exchanged with the host in return for a carbon source [31].

Despite its abundance in the atmosphere, nitrogen is often the most limiting nutrient for plant growth. In order for nitrogen to be assimilated by plants, it must be ‘fixed’ or combined in the form of ammonium (NH4+) or nitrate (NO3) ions [32]. However, ammonium at high concentrations can be toxic for plants [33]. Animals receive the required nitrogen they need for metabolism, growth and reproduction by the consumption of living or dead organic matter containing molecules composed partially of nitrogen.

In most ecosystems, nitrogen is stored primarily in living and dead organic matter. This organic nitrogen is converted into inorganic forms by decomposition. Organisms that serve as decomposers are usually found in the upper soil layer and chemically modify organic nitrogen. Usually, nitrogen is returned to the soil via animal waste or from the output of decomposers, in the form of ammonia (NH3) which is absorbed on to the surfaces of clay particles in the soil. Subsequently, NH4+ is released from the colloids by way of cation exchange and oxidized by autotrophic bacteria first to NO2 and then to NO3. However, since NO3 is very soluble, it is easily lost from the soil system by leaching. Some of this leached nitrate flows through the hydrologic system until it reaches the oceans, where it can be returned to the atmosphere by denitrification. The gaseous products of denitrification (N2, NO or N2O) diffuse into the atmosphere.

Molecular approaches have been applied to the study of the structure of microbial communities involved in nitrogen cycling in ecosystems as diverse as soil, estuarine and coastal wetlands, oceans, forests, cultivars and extreme environments (salt marshes, acidic rivers, salted or frosty soils and arctic tundra) [34,35].

Anthropogenic impacts on the nitrogen cycle

During the 20th Century, human activities, particularly the chemical reduction of nitrogen on an industrial scale to make synthetic fertilizers and the combustion of fossil fuels, have had an increasingly significant effect on the global nitrogen cycle [3]. Unsurprisingly, there is a disproportionate impact by human populations in developed countries, where vehicle emissions and industrial agriculture are most prevalent [6]. These influence climate change, the chemistry of the atmosphere, human health and the ecological functioning of natural ecosystems, especially aquatic systems and soils where nitrogen concentrations are increasing, causing eutrophication of lakes or rivers and oceanic dead zones through algal bloom-induced hypoxia [7].

The nitrogen compounds resulting from human activities that have the greatest impact on the environment are the following.

(i) Enhanced NO and N2O emissions from fertilized soils due to denitrification. These gases are also produced through biomass burning, cattle and feedlots, fossil fuel combustion and other industrial sources. N2O along with carbon dioxide (CO2) and methane (CH4) are the three most important greenhouse gases. Not only does N2O have a 300-fold greater global warming potential than CO2, but also its atmospheric loading is increasing by 0.25% each year. As a consequence, it is essential that strategies to mitigate climate change include the reduction of N2O emissions [5].

In addition, both N2O and NO have deleterious effects on the stratosphere, where they act as catalysts in the destruction of atmospheric ozone. Indeed, it has been reported that N2O is currently is the single most important ozone-depleting emission and is expected to remain the largest throughout the 21st Century [36]. Limiting future anthropogenic N2O emissions would not only allow the recovery of the depleted ozone layer, but also reduce climate change [37].

(ii) Excess NO3 and NO2 derived from fertilizers are leached from soils and enter the groundwater. At concentrations of <5 mM, NO2 is toxic for most micro-organisms [38], and considerably lower concentrations pose a threat to aquatic invertebrates [39]. Elevated levels of nitrate in drinking water is a known risk factor for methaemoglobinaemia (a potential cause of blue baby syndrome) [8] and colon cancer [9]. The currently accepted, and costly to maintain, safe limit for nitrate in drinking water is 10 p.p.m. in the U.S.A. and 11.3 p.p.m. in the EU (European Union), but the requirement to maintain such stringent limits is contentious [40,41].

(iii) NH3 in the atmosphere has tripled as the result of human activities. It acts as an aerosol, decreasing air quality and clinging on to water droplets [42].

(iv) The addition of nitrogen to ecosystems reduces biodiversity and thereby leads to loss of ecosystem function. An excessive addition of nitrogen compounds to the environment was perhaps first noted through the eutrophication of water bodies that results in a loss of species diversity [6].

New challenges for nitrogen-cycle research

Although some aspects of the biochemistry and molecular biology of the nitrogen cycle are well understood, our knowledge is far from complete. Recent environmental metagenomic studies have provided evidence of novel enzyme activities and metabolic pathways [29,43] that will need to be understood at the molecular level if they are to be fully exploited. There is also the challenge of applying what we have learnt to the management strategies and policy decisions that will shape the environment of the 21st Century.

Taking just one example: at the molecular level, denitrification is perhaps the best understood of the processes that contribute to the nitrogen cycle, yet there are gaps in our knowledge, which makes it difficult to generate a robust mathematical model that can describe the metabolism of a single organism in pure culture [44]. Such a model scaled up to the field could inform management strategies that might mitigate N2O emissions and make more efficient use of synthetic fertilizers [5]. Paradoxically, some strategies based on biofuel production designed to mitigate the effects of CO2 release from fossil fuels actually lead to increases in global warming potential and ozone depletion because of the increased requirement for artificial fertilizers [45].

Footnotes

  • Enzymology and Ecology of the Nitrogen Cycle: A Biochemical Society Focused Meeting held at University of Birmingham, U.K., 15–17 September 2010. Organized and Edited by Jeff Cole (University of Birmingham, U.K.), Rosa María Martínez-Espinosa (University of Alicante, Spain), David Richardson (University of East Anglia, Norwich, U.K.) and Nick Watmough (University of East Anglia, Norwich, U.K.).

References

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