Enzymology and Ecology of the Nitrogen Cycle

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 Marı́a Martı́nez-Espinosa (University of Alicante, Spain), David Richardson (University of East Anglia, Norwich, U.K.) and Nick
Watmough (University of East Anglia, Norwich, U.K.).

Enzymology and ecology of the nitrogen cycle

Rosa Marı́a Martı́nez-Espinosa*1 , Jeffrey A. Cole†, David J. Richardson‡ and Nicholas J. Watmough‡
*Department of Agricultural Chemistry and Biochemistry, Science Faculty Phase II, University of Alicante. 03690 San Vicente del Raspeig, Alicante, Spain,
†School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, U.K. and ‡Centre for Molecular and Structural Biochemistry, School of
Biological Sciences, University of East Anglia, Norwich, Norfolk NR4 7TJ, U.K.

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.

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 (NH2 OH), nitrate (NO3 − ), nitrite (NO2 − ),
nitric oxide (NO), nitrous oxide (N2 O) 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
Key words: ammonium, nitrate, nitric oxide, nitrite, nitrogen cycle, nitrous oxide.
1
To whom correspondence should be addressed (email rosa.martinez@ua.es).
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]. Con-
sequently, 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

Biochem. Soc. Trans. (2011) 39, 175–178; doi:10.1042/BST0390175 

C The Authors Journal compilation 
C 2011 Biochemical Society 175
176 Biochemical Society Transactions (2011) Volume 39, part 1

cycle, both prokaryotic and plant nitrogen metabolism have culture that couples the anaerobic oxidation of methane with
become major areas of research over the last few years. the reduction of nitrite to dinitrogen has pointed to the
The global biochemical nitrogen cycle includes redox existence of previously undescribed enzyme activities [29].
reactions used for assimilatory purposes or in respiratory
pathways for energy conservation. The assimilatory reactions
include nitrogen fixation (N2 conversion into ammonia by
Ecology of the nitrogen cycle
free-living or symbiotic bacteria) [11] and assimilatory nitrate
The store of nitrogen found in the atmosphere is about one
reduction (the reduction of nitrate to ammonium) [12,13].
million times larger than the total nitrogen contained in living
Both pathways produce ammonium which is incorporated
organisms. Other major stores of nitrogen include organic
into carbon skeletons for growth. Apart from the respiratory
matter in soil and the oceans. Almost all of the nitrogen
reduction of nitrate to ammonia that is widespread among
found in any terrestrial ecosystem originally came from the
anaerobic fermentative bacteria, there are two major energy-
atmosphere. Although significant amounts enter the soil in
generating processes: nitrification (soil-living bacteria and
rainfall or through the effects of lightning [30], the majority of
other nitrifying bacteria oxidize ammonia to nitrate) [14],
nitrogen is biochemically fixed within the soil by specialized
and denitrification (anaerobic or facultative anaerobic
micro-organisms that live symbiotically with plants. The
microbes reduce nitrate to N2 or other nitrogen gases
bacteria live in a specialized microenvironment provided by
under anaerobic conditions) [15]. To complete the global
the plant (root nodules) in which they can reduce nitrogen,
nitrogen cycle, two other pathways are carried out only by
some of which is exchanged with the host in return for a
micro-organisms: ammonification (many bacteria and fungi
carbon source [31].
degrade organic matter, releasing fixed nitrogen for reuse
Despite its abundance in the atmosphere, nitrogen is
by other organisms), and anammox (anaerobic ammonium
often the most limiting nutrient for plant growth. In
oxidation that produces N2 by reducing nitrite and oxidizing
order for nitrogen to be assimilated by plants, it must be
ammonium) [16,17].
‘fixed’ or combined in the form of ammonium (NH4 + )
As a result of this biogeochemical cycle, the percentage
or nitrate (NO3 − ) ions [32]. However, ammonium at high
of nitrogen in the atmosphere remains constant: nitrogen is
concentrations can be toxic for plants [33]. Animals receive
continually released into the air by the action of denitrifying
the required nitrogen they need for metabolism, growth and
bacteria and continually returned to the cycle through the
reproduction by the consumption of living or dead organic
action of nitrogen fixing micro-organisms, lightning and
matter containing molecules composed partially of nitrogen.
industrial production of artificial fertilizer.
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
Enzymology of the nitrogen cycle as decomposers are usually found in the upper soil layer
The simplest scheme to describe a complete nitrogen cycle
and chemically modify organic nitrogen. Usually, nitrogen
involves five reduction and three oxidation reactions [18].
is returned to the soil via animal waste or from the output
In reality, one ecosystem or even a single micro-organism
of decomposers, in the form of ammonia (NH3 ) which is
that can, for example, reduce nitrate under both aerobic and
absorbed on to the surfaces of clay particles in the soil.
anaerobic conditions is rather complicated. Many enzyme-
Subsequently, NH4 + is released from the colloids by way
catalysed reactions are involved, all of which are highly
of cation exchange and oxidized by autotrophic bacteria first
regulated. Understanding the structure and mechanism of the
to NO2 − and then to NO3 − . However, since NO3 − is very
metal-containing enzymes that catalyse the redox reactions of
soluble, it is easily lost from the soil system by leaching.
the nitrogen cycle [19,20] has been a major research topic over
Some of this leached nitrate flows through the hydrologic
the last two decades. Considerable progress has been made
system until it reaches the oceans, where it can be returned to
with some enzymes, such as nitrite reductases [21,22], but
the atmosphere by denitrification. The gaseous products of
significant challenges remain if we are to fully understand
denitrification (N2 , NO or N2 O) diffuse into the atmosphere.
bacterial NO reduction [23] and the assembly of the N2 O
Molecular approaches have been applied to the study of
reductase [24], while the enzymology of anammox remains
the structure of microbial communities involved in nitrogen
poorly described.
cycling in ecosystems as diverse as soil, estuarine and coastal
Progress in understanding the biochemistry of the nitrogen
wetlands, oceans, forests, cultivars and extreme environments
cycle has been complemented by the availability of the
(salt marshes, acidic rivers, salted or frosty soils and arctic
complete genome sequences of hundreds of micro-organisms.
tundra) [34,35].
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- Anthropogenic impacts on the nitrogen
cycle proteins has allowed the evolution of this important cycle
cycle to be analysed across the three domains of life [27,28]. During the 20th Century, human activities, particularly the
More recently, metagenomic sequencing of an enrichment chemical reduction of nitrogen on an industrial scale to


C The Authors Journal compilation 
C 2011 Biochemical Society

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 N2 O 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. N2 O along with carbon dioxide
(CO2 ) and methane (CH4 ) are the three most important
greenhouse gases. Not only does N2 O 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 N2 O emissions [5].
In addition, both N2 O 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 N2 O
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 N2 O
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 concentra-
tions 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 drink-
ing 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
Enzymology and Ecology of the Nitrogen Cycle 177
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, denitri-
fication 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 N2 O 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].
References
1 Richardson, D.J. and Watmough, N.J. (1999) Inorganic nitrogen
metabolism in bacteria. Curr. Opin. Chem. Biol. 3, 207–219
2 Canfield, D.E., Glazer, A.N. and Falkowski, P.G. (2010) The evolution and
future of Earth’s nitrogen cycle. Science 330, 192–196
3 Galloway, J.N., Townsend, A.R., Erisman, J.W., Bekunda, M., Cai, Z.,
Freney, J.R., Martinelli, L.A., Seitzinger, S.P. and Sutton, M.A. (2008)
Transformation of the nitrogen cycle: recent trends, questions, and
potential solutions. Science 320, 889–892
4 Duce, R.A., LaRoche, J., Altieri, K., Arrigo, K.R., Baker, A.R., Capone, D.G.,
Cornell, S., Dentener, F., Galloway, J., Ganeshram, R.S. et al. (2008)
Impacts of atmospheric anthropogenic nitrogen on the open ocean.
Science 320, 893–897
5 Richardson, D., Felgate, H., Watmough, N., Thomson, A. and Baggs, E.
(2009) Mitigating release of the potent greenhouse gas N2 O from the
nitrogen cycle: could enzymic regulation hold the key? Trends
Biotechnol. 27, 388–397
6 Socolow, R.H. (1999) Nitrogen management and the future of food:
lessons from the management of energy and carbon. Proc. Natl. Acad.
Sci. U.S.A. 96, 6001–6008
7 Howarth, R.W. (2004) Human acceleration of the nitrogen cycle: drivers,
consequences, and steps toward solutions. Water Sci. Technol. 49, 7–13
8 Greer, F.R. and Shannon, M. (2005) Infant methemoglobinemia: the role
of dietary nitrate in food and water. Pediatrics 116, 784–786
9 Van Grinsven, H.J., Rabl, A. and de Kok, T.M. (2010) Estimation of
incidence and social cost of colon cancer due to nitrate in drinking water
in the EU: a tentative cost-benefit assessment. Environ. Health 9, 58
10 Richardson, D.J. (2001) Introduction: nitrate reduction and the nitrogen
cycle. Cell. Mol. Life Sci. 58, 163–164
11 Newton, W.E. (2007) Physiology, biochemistry and molecular biology of
nitrogen fixation. In Biology of the Nitrogen Cycle (Bothe, H., Ferguson,
S.J. and Newton, W.E., eds), pp. 109–129, Elsevier, Amsterdam
12 Moreno-Vivian, C. and Flores, E. (2007) Nitrate assimilation in bacteria. In
Biology of the Nitrogen Cycle (Bothe, H., Ferguson, S.J. and Newton,
W.E., eds), pp. 263–282, Elsevier, Amsterdam
13 Tischner, R. and Kaiser, W. (2007) Nitrate assimilation in plants. In
Biology of the Nitrogen Cycle (Bothe, H., Ferguson, S.J. and Newton,
W.E., eds), pp. 283–301, Elsevier, Amsterdam
14 Ferguson, S.J., Richardson, D.J. and Van Spanning, R.J.M. (2007)
Biochemistry and molecular biology of nitrification. In Biology of the
Nitrogen Cycle (Bothe, H., Ferguson, S.J. and Newton, W.E., eds),
pp. 209–222, Elsevier, Amsterdam
15 Van Spanning, R.J.M., Richardson, D.J. and Ferguson, S.J. (2007)
Introduction to the biochemistry and molecular biology of denitrification.
In Biology of the Nitrogen Cycle (Bothe, H., Ferguson, S.J. and Newton,
W.E., eds), pp. 3–20, Elsevier, Amsterdam

C The Authors Journal compilation 
C 2011 Biochemical Society
178 Biochemical Society Transactions (2011) Volume 39, part 1
16 Jetten, M.S., Niftrik, L., Strous, M., Kartal, B., Keltjens, J.T. and Op den
Camp, H.J. (2009) Biochemistry and molecular biology of anammox
bacteria. Crit. Rev. Biochem. Mol. Biol. 44, 65–84
17 Op den Camp, H.J.M., Jetten, M.S.M. and Strous, M. (2007) Anammox. In
Biology of the Nitrogen Cycle (Bothe, H., Ferguson, S.J. and Newton,
W.E., eds), pp. 245–262, Elsevier, Amsterdam
18 Ferguson, S.J. (1998) Nitrogen cycle enzymology. Curr. Opin. Chem. Biol.
2, 182–193
19 Berks, B.C., Ferguson, S.J., Moir, J.W.B. and Richardson, D.J. (1995)
Enzymes and associated electron transport systems that catalyse the
respiratory reduction of nitrogen oxides and oxyanions. Biochim.
Biophys. Acta 1232, 97–173
20 Butler, C.S. and Richardson, D.J. (2005) The emerging molecular structure
of the nitrogen cycle: an introduction to the proceedings of the 10th
annual N-cycle meeting. Biochem. Soc. Trans. 33, 113–118
21 Antonyuk, S.V., Strange, R.W., Sawers, G., Eady, R.R. and Hasnain, S.S.
(2005) Atomic resolution structures of resting-state, substrate- and
product-complexed Cu-nitrite reductase provide insight into catalytic
mechanism. Proc. Natl. Acad. Sci. U.S.A. 102, 12041–12046
22 Rinaldo, S., Arcovito, A., Giardina, G., Castiglione, N., Brunori, M. and
Cutruzzola, F. (2008) New insights into the activity of Pseudomonas
aeruginosa cd 1 nitrite reductase. Biochem. Soc. Trans. 36, 1155–1159
23 Zumft, W.G. (2005) Nitric oxide reductases of prokaryotes with emphasis
on the respiratory, heme-copper oxidase type. J. Inorg. Biochem. 99,
194–215
24 Zumft, W.G. and Kroneck, P.M. (2007) Respiratory transformation of
nitrous oxide (N2 O) to dinitrogen by Bacteria and Archaea. Adv. Microb.
Physiol. 52, 107–227
25 Constantinidou, C., Hobman, J.L., Griffiths, L., Patel, M.D., Penn, C.W.,
Cole, J.A. and Overton, T.W. (2006) A reassessment of the FNR regulon
and transcriptomic analysis of the effects of nitrate, nitrite, NarXL, and
NarQP as Escherichia coli K12 adapts from aerobic to anaerobic growth.
J. Biol. Chem. 281, 4802–4815
26 Overton, T.W., Whitehead, R., Li, Y., Snyder, L.A., Saunders, N.J., Smith, H.
and Cole, J.A. (2006) Coordinated regulation of the Neisseria
gonorrhoeae-truncated denitrification pathway by the nitric
oxide-sensitive repressor, NsrR, and nitrite-insensitive NarQ-NarP. J. Biol.
Chem. 281, 33115–33126
27 Brown, J.R. and Doolittle, W.F. (1997) Archaea and the
prokaryote-to-eukaryote transition. Microbiol. Mol. Biol. Rev. 61,
456–502
28 Brown, J.R., Masuchi, Y., Robb, F.T. and Doolittle, W.F. (1994)
Evolutionary relationships of bacterial and archaeal glutamine
synthetase genes. J. Mol. Evol. 38, 566–576
29 Ettwig, K.F., Butler, M.K., Le Paslier, D., Pelletier, E., Mangenot, S.,
Kuypers, M.M., Schreiber, F., Dutilh, B.E., Zedelius, J., de Beer, D. et al.
(2010) Nitrite-driven anaerobic methane oxidation by oxygenic bacteria.
Nature 464, 543–548
30 Rakov, V.A. and Uman, M.A. (2007) Lightning: Physics and Effects,
Cambridge University Press, Cambridge

C The Authors Journal compilation 
C 2011 Biochemical Society
31 Stacey, G. (2007) The Rhizobium−legume nitrogen fixing symbiosis. In
Biology of the Nitrogen Cycle (Bothe, H., Ferguson, S.J. and Newton,
W.E., eds), pp. 147–163, Elsevier, Amsterdam.
32 Mancinelli, R.L. (1996) The nature of nitrogen: an overview. Life Support
Biosph. Sci. 3, 17–24
33 Britto, D.T. and Kronzuker, H.J. (2002) NH4 + toxicity in higher plants: a
critical review. J. Plant Physiol. 159, 567–584
34 Cuhel, J., Simek, M., Laughlin, R.J., Bru, D., Cheneby, D., Watson, C.J. and
Philippot, L. (2010) Insights into the effect of soil pH on N2 O and N2
emissions and denitrifier community size and activity. Appl. Environ.
Microbiol. 76, 1870–1878
35 Taroncher-Oldenburg, G., Griner, E.M., Francis, C.A. and Ward, B.B. (2003)
Oligonucleotide microarray for the study of functional gene diversity in
the nitrogen cycle in the environment. Appl. Environ. Microbiol. 69,
1159–1171
36 Lassey, D. and Harvey, N. (2007) Nitrous oxide: the serious side of
laughing gas. Water Atmosph. 15, 10–11
37 Ravishankara, A.R., Daniel, J.S. and Portmann, R.W. (2009) Nitrous oxide
(N2 O): the dominant ozone-depleting substance emitted in the 21st
century. Science 326, 123–125
38 Shen, Q.R., Ran, W. and Cao, Z.H. (2003) Mechanisms of nitrite
accumulation occurring in soil nitrification. Chemosphere 50, 747–753
39 Alonso, A. and Camargo, J.A. (2006) Toxicity of nitrite to three species of
freshwater invertebrates. Environ. Toxicol. 21, 90–94
40 Powlson, D.S., Addiscott, T.M., Benjamin, N., Cassman, K.G., de Kok, T.M.,
van Grinsven, H., L’Hirondel, J.L., Avery, A.A. and van Kessel, C. (2008)
When does nitrate become a risk for humans? J. Environ. Qual. 37,
291–295
41 van Grinsven, H.J., Ward, M.H., Benjamin, N. and de Kok, T.M. (2006)
Does the evidence about health risks associated with nitrate ingestion
warrant an increase of the nitrate standard for drinking water? Environ.
Health 5, 26
42 Harper, L.A., Flesch, T.K. and Wilson, J.D. (2010) Ammonia emissions from
broiler production in the San Joaquin Valley. Poult. Sci. 89, 1802–1814
43 Lucker, S., Wagner, M., Maixner, F., Pelletier, E., Koch, H., Vacherie, B.,
Rattei, T., Damste, J.S., Spieck, E., Le Paslier, D. and Daims, H. (2010) A
Nitrospira metagenome illuminates the physiology and evolution of
globally important nitrite-oxidizing bacteria. Proc. Natl. Acad. Sci. U.S.A.
107, 13479–13484
44 Molstad, L., Dörsch, P. and Bakken, L.R. (2007) Robotized incubation
system for monitoring gases (O2 , NO, N2 O, N2 ) in denitrifying cultures. J.
Microbiol. Methods 71, 202–211
45 Hill, J., Nelson, E., Tilman, D., Polasky, S. and Tiffany, D. (2006) From the
cover: environmental, economic, and energetic costs and benefits of
biodiesel and ethanol biofuels. Proc. Natl. Acad. Sci. U.S.A. 103,
11206–11210
Received 19 November 2010
doi:10.1042/BST0390175