Regulation of denitrification at the cellular level: a clue to the understanding of N2O emissions from soils.

2012·0.62 MB·English

by  Bakken, Lars R.

Phil. Trans.R.Soc. B(2012) 367, 1226–1234
doi:10.1098/rstb.2011.0321
Research
Regulation of denitrification at the cellular
level: a clue to the understanding of N O
2
emissions from soils
Lars R. Bakken1,*, Linda Bergaust1, Binbin Liu2 and A˚sa Frostega˚rd2
1Department of Plant and Environmental Sciences, and 2Department of Chemistry, Biotechnology
and Food Sciences, Norwegian University of Life Sciences, PO Box 5003, 1432 Aas, Norway
Denitrifying prokaryotes use NO as terminal electron acceptors in response to oxygen depletion.
x
TheprocessemitsamixtureofNO,N OandN ,dependingontherelativeactivityoftheenzymes
2 2
catalysing the stepwise reduction of NO2 to N O and finally to N . Cultured denitrifying
3 2 2
prokaryotes show characteristic transient accumulation of NO2, NO and N O during transition
2 2
from oxic to anoxic respiration, when tested under standardized conditions, but this character
appears unrelated to phylogeny. Thus, although the denitrifying community of soils may differ in
their propensity to emit N O, it may be difficult to predict such characteristics by analysis of the
2
community composition. A common feature of strains tested in our laboratory is that the relative
amounts of N O produced (N O/(N þN O) product ratio) is correlated with acidity, apparently
2 2 2 2
owing to interference with the assembly of the enzyme N O reductase. The same phenomenon
2
was demonstrated for soils and microbial communities extracted from soils. Liming could be a
way to reduce N O emissions, but needs verification by field experiments. More sophisticated
2
ways to reduce emissions may emerge in the future as we learn more about the regulation of
denitrification at the cellular level.
Keywords: nitrous oxide; soil; physiology; regulation; mitigation
1. DENITRIFICATION IN SOILS: A MAJOR global warming and is recognized as the major factor
SOURCE OF ATMOSPHERIC N O causing destruction of stratospheric ozone [3].
2
Human activities have more than doubled the annual TheglobalN Oemissionsfromcultivatedsoilshave
2
input of reactive nitrogen to the biosphere compared been estimated at 3.3Tg N Oyr21 [4], which is
2
with prehistoric rates. This anthropogenic reactive around 50 per cent of the estimated global anthropo-
N is primarily fertilizer N, biologically fixed N (in genic N O emission. A substantial part of the
2
legume crops) and NO from combustion. The remaining anthropogenic N O emissions (from water
x 2
ongoingamplificationoftheglobalNcyclehasalready bodies and uncultivated areas) are driven by nitrogen
alteredthebiogeochemicalprocessesofnaturalecosys- losses from agroecosystems as well [5]. There is thus
tems, their trophic dynamics and biological diversity littledoubtthatterrestrialhabitats(bothcultivatedand
[1]. The ultimate fate of anthropogenic nitrogen is a uncultivated) are one of the most important sources of
return to the atmosphere, either as N , N O or NO, atmospheric N O. Denitrification is probably a much
2 2 2
which are the gaseous products of microbial red/ morepotentN Osourcethannitrification,asindicated
2
ox-transformations of mineral nitrogen. The N-gas bythelowN O/NO2productstoichiometryofnitrifica-
2 3
product-stoichiometry of these processes is controlled tion [6]. Tracer studies suggesting a major role of
bytheecologyandregulatorybiologyoftheorganisms nitrification may possibly have confounded coupled
involved, as modulated by environmental factors. nitrification–denitrificationvianitrite[7].
A better understanding and quantification of these Fewtoolsareavailabletoreducetheemissionsfrom
processes is urgently needed to improve our chances natural ecosystems, apart from reducing the inputs of
to minimize N Oemissions [2]. The currentaccumu- anthropogenic reactive nitrogen. The effect of such
2
lation of N O in the atmosphere accounts for reductions may be slow, however, owing to the long
2
approximately 9 per cent of estimated anthropogenic residence time of reactive nitrogen (as biomass and
soil organic N) within the systems [8]. Cultivated
areas, on the other hand, are intensively managed by
drainage, cropping, tillage, liming, fertilization and
*Authorforcorrespondence([email protected]). use of agrochemicals. These operations should hold
a potential for reducing the N O emissions, provided
Onecontributionof12toaTheoMurphyMeetingIssue‘Nitrous 2
oxide:theforgottengreenhousegas’. that we know how they affect the activity of the
1226 Thisjournalisq2012TheRoyalSociety
Regulation at cellular level L. R. Bakken et al. 1227
organisms responsible for N O production in the soil. drainageofpaddyricesoils(drainageisoftenincluded
2
DespiteseveraldecadesofresearchonN Oemissions, intheannualmanagementofsuchfields)willenhance
2
however,few(ifany)practicalwaystoachievesubstan- the N O emission [10]; in farmed organic soils, the
2
tial reductions of N O emissions have been invented. lowering of the water table (i.e. drainage) may or
2
Whyistheresolittleprogress?Howcanweimprove may not reduce N O emissions depending on other
2
ourefforts? We suspect that one of the reasons for the factors [11], whereas adequate artificial drainage to
current shortcoming of the research on N O emission sustain a low water table depth throughout the whole
2
is that there has been too little cross-talk between dis- year appears to reduce N O emissions from grassland
2
ciplines. Great progress has been achieved from soils[12].Anotherexampleistillage,whichmayresult
studies of the biochemistry, regulatory biology and in higher or lower annual N O emissions, apparently
2
physiology of denitrifying prokaryotes over the last depending on the climate and possibly a number of
two decades, driven by molecular biology. Yet, too soil variables ([13] and references therein).
little of this knowledge has reached the agronomists/ Athirdshortcomingofnearlyallfieldexperimentsis
biogeochemists/earth scientists who have made great that the N O/N productratio hasnot been measured,
2 2
progress within their own fields. Most experiments primarilybecauseithasproveddifficulttoobtainreliable
within each of the disciplines mentioned are designed field measurements of N production [14]. The acet-
2
tosolve‘disciplinary’problems,byscientistswithonly ylene inhibition method may be used, but is likely to
rudimentary understanding of how adjacent disci- underestimate denitrification owing to diffusion limit-
plines think and work, and their recent progress. We ation [15] and acetylene-catalyzed oxidation of NO
believethatbetterinteractionanddiscussionsbetween [16]. Tracer-based measurements are possible [17],
the disciplines hold a potential for progress in the buthavenotbeenusedmuchbecausetheyarecompli-
search for ways to mitigate N O emission. cated, demanding continuous monitoring of both gas
2
The present paper is an attempt to illustrate this emissions and the tracer (15N) concentration in the
potential, by reviewing recent progress in a series of nitrate/nitrite pool of the soil. The scarcity of reliable
experimental approaches dedicated to understanding measurementsofN productioninfieldexperiments is
2
the mechanisms at the cellular level which control the most unfortunate, since the N O/N product ratios
2 2
N O/(N þN O) product ratio of denitrification. This shouldbeatargetfordesigningmitigationoptions[2].
2 2 2
ratio is of utmost importance for the earth sciences in ThepotentialbenefitofmeasuringbothN andN O
2 2
theirattempttolinkN Oemissionstothecurrentampli- productionisillustratedbytwostudiesofintactsoilsfrom
2
fication of the nitrogen cycle. Before diving into the field experiments conducted by Zaman et al. [18,19],
microbiologicalinvestigations,weneedtobrieflyrecapi- where they quantified both N O and N production
2 2
tulate some research on N O emissions within the (the latter by the acetylene inhibition technique). They
2
earthsciences. found that liming invariably reduced the N O/N pro-
2 2
duct ratio, and the effect on net N O emission was
2
either nil [18] or positive (higher emission from limed
2. FIELD EXPERIMENTS soil [19]). Their quantification of N production by
2
N Oemissionsfromagriculturalsoilshavebeenstudied the acetylene inhibition method may have been biased
2
intensively in numerous field trials on all continents, forthereasonmentionedearlier(lackofsteady-statecon-
reviewed and analysed thoroughly by Stehfest & ditionsowingtorestricteddiffusion,andNOscavenging
Bouwman [4]. Excess fertilizer N doses result in high by acetylene). Nevertheless, their observation of an
emissions, but N O emissions correlate only poorly alteredN O/N productratioisprobablyvalidsincewe
2 2 2
with the fertilizer N level when within realistic ranges. mayassumethatthebiasisthesameinalltreatments.
Numerous other factors are involved in controlling the
N O emission from soils, which contribute to the huge
2
spatio-temporal variation in emissions in field trials 3. ECOSYSTEM MODELLING
[4,9]. An unfortunate consequence of the spatio- BiogeochemicalmodellingofdenitrificationanditsN O
2
temporal variation is that the power of statistical tests production are based on relatively crude models of the
ofsuchexperimentsislow,i.e.althoughtheexperimen- process and its regulation at the cellular level. Much
taltreatments mayhavearealeffect,there isa highrisk effort has been invested in modelling the heat-, water-
oferroneouslyconcludingthattherewasnoeffect (type and not the least oxygen-transport within the soil
II error). A number of management effects may have matrix [20]. Less effort has been invested in refining
been erroneously rejected for this reason. A simple cal- the modelling of denitrifying community phenotype,
culation of the confidence interval of the difference whichisa‘blackbox’inmostmodels,basedonrelatively
between contrasting treatments could reveal the pitfall; antique parameters forenzyme and growth kinetics. In
i.e. the substantial effect of a management could have addition,allsoilsareassumedtoharbourthesamecom-
existedwithoutbeingdetectedasstatisticallysignificant. munity phenotype [9,21]. It appears bluntly wrong to
Thisisoftenneglected,possiblybecauseitisfrustrating assume that all community phenotypes are equal, as
toilluminatethelowstatisticalpoweroftheexperiment. judged from investigations of community phenotypes
Another problem with field trials is that the obser- of contrasting soils [22,23]. Despite these (and other)
vations tend to be ‘anecdotal’ because the effects of gross simplifications, the simulated annual N O emis-
2
specific operations on N O emissions are likely to be sions are often in reasonable agreement with measured
2
conditional, i.e. dependent on several other variables values for contrasting soils [24] and for contrasting
suchassoiltype,climateandothermanagementoper- soilmanagementscenarios[25],althoughthetemporal
ations. Drainage of the soils is a good example: variationisstillachallenge[20,24].
Phil.Trans.R.Soc.B(2012)
1228 L. R. Bakken et al. Regulation at cellular level
(a) (b)
2.5
nirS
1 2.0 norB
–ell nosZ
s c 1.5 nosZ/norB: 6.6 nosZ/norB: 6.4
pie nosZ/norS: 1.1 nosZ/norS: 2.2
o
c
A 1.0
N
R
m
0.5
0 20 40 60 80 0 20 40 60 80
time (h) time (h)
Figure 1. Transcription of nirS, norB and nosZ in Paracoccus denitrificans in response to oxygen depletion when grown in
Sistrom’s medium with 34mM succinate as the main C source. Results are shown for two pH levels ((a) pH 7; (b) pH 6).
The relative transcription of nosZ is expressed by nosZ/norB and nosZ/nirS ratios in each panel, based on the four highest
transcript numbersrecordedforeach gene(from Bergaust etal.[37]).
Simplificationsarenecessaryinbiogeochemicaleco- of denitrifying prokaryotes, with a deliberate focus
system modelling, but it seems likely that the existing on their ability to express N O reductase. We have
2
modelscouldbeimprovedthroughadialoguebetween dedicated ourselves to work along these lines, and
the modellers and the research community working the rest of this paper is a first attempt to summarize
with the genetic and physiological basis of denitrifica- some of our findings.
tion. This is particularly true if the purpose of the
modelling is to design ‘mitigation measures’, i.e. soil
and fertilization routines that reduce the N O emis- 4. N O FROM DENITRIFICATION DEPENDS ON
2 2
sions. As stated previously, a reduction in the N O/ REGULATION AT THE CELLULAR LEVEL
2
(N þN O) product ratio of denitrification should be Denitrification is the stepwise reduction of NO2
2 2 3
a target when searching for mitigation measures. The throughNO2,NO,N OtoN ,drivenbyfourreductase
2 2 2
existing models are possibly inadequate for this pur- enzymes NAR/NAP, NIR, NOR and N OR, respect-
2
pose, although this is unknown because we lack ively. This enables the organisms to sustain respiratory
reliable datafor N production in field experiments to metabolismduringoxygenlimitation,withNO asterm-
2 x
test their predictions. It would be arrogant (but poss- inal electron acceptors. The denitrification proteome
ibly legitimate) to claim that when a nearly perfect (NAR, NIR, NOR and N OR plus several other
2
match is found between measured and simulated proteins)issynthesizedinresponsetooxygendepletion,
annual N O emissions, the biogeochemical model is and is blocked by high oxygen concentrations (both
2
rightforanumberofwrongreasons.Itislessarrogant transcriptional and post-transcriptional control [33]).
(but fully legitimate) to claim that if used to simulate The transcription of the genes coding for the indi-
phenomena for which the models are not rigorously vidual reductases is controlled by a network of
tested,their predictionsareratherhypothetical. transcriptional regulators and ancillary factors
Ourimpressionisthatfieldexperimentsandmodel- [34,35] which respond to intra- and extracellular sig-
ling of N O emissions have not made much progress nals among which are oxygen and N-oxides (NO and
2
regarding mitigation of N O emissions. This is NO2). Although important ‘nuts and bolts’ of this
2 2
reflected in several reviews of available mitigation regulatory network have been unravelled for a few
options, which generally recommend operations that model strains, such as Paracoccus denitrificans, much
(i) secure synchronization of mineralization with the remains to be discovered before we are able to fully
needformineralNbygrowingcropsand(ii)minimize understand the phenotypic response. For instance,
the mineral N concentrations in the soil during off- we have found that phenotypic responses of various
season [26–31], which are no more than the general mutantsofP.denitrificanssuggestthatthetranscription
recommendations for good agronomic practice to of nar requires both FnrP and NarR (dual control),
minimize nitrate leaching [32]. A perpetuation of while the transcription of nosZ (coding for N OR) is
2
field experiments and modelling of N O emissions equally effective with only FnrP (in response to
2
along the same track will probably not result in sub- oxygen depletion) or NNR (responding to NO) [36].
stantial progress regarding mitigations. That is not to Another conspicuous finding regarding the regulation
say that such activities should be terminated. Field in P. denitrificans is that N OR is expressed much ear-
2
emission data are extremely important for several lier than NIR and NOR (and possibly NAR), as
obvious reasons, and any hypothetical mitigation illustrated in figure 1. Moreover, only a fraction of
must be tested in field experiments. We areconvinced the cells are actually able to express NIR and NOR
thatifthereexistnovelwaystoreduceN Oemissions, in due time before all the oxygen has been depleted
2
such mitigation options are more likely to be found [37,38].Wehypothesizethatthisisduetoastochastic
through studies of the ecology and regulatory biology initiationofthetranscriptionofnirandnor,whichthen
Phil.Trans.R.Soc.B(2012)
Regulation at cellular level L. R. Bakken et al. 1229
[O ] µM that for P. denitrificans shown in figure 2 [40]. One of
uid, 80 [N2O] nM the strains lacked nosZ and produced 100 per cent
d [NO] nM in liq–1µmol flask) 4600 N2 µmol svaNNterrO2yraOyi32ng,sseiwtmnowheiasilNlasert2roto.thobeAmuPss.aectkdvoceeemonnnaimtotrriotcofihonlcemaorfnfepssaltNert(aut5Oeirn–esa3dto5ehnfnaniadtaMnrlailofilmiltcnhatohettliehaoTernnhleleaicfqvrueeuoeslmrissda-,
an (2 phase). They deviated grossly, however, regarding the
M N 20 transient accumulation of both NO22 and N2O. Some
] µ2 of the strains reduced all NO32 to NO22 before expres-
O sing NIR and NOR, wheras others accumulated
[
negligible amounts of NO2. The strains were also
0 20 40 60 2
time (h) different regarding the transient accumulation of
N O, ranging from 0.06 to 5 per cent of all NO2-N
2 3
Figure 2. The typical denitrification phenotype of
finally reduced to N . The results are summarized in
P. denitrificans in response to oxygen depletion in a 50ml 2
figure 4, together with their phylogenetic relationship
batch of Sistrom’s medium with 2mM NO2 and pH¼7.
3 based on 16S rDNA.
The graph shows O and NO concentration in the liquid
2 There exists a plethora of similar studies where the
(asmMandnM,respectively)andcumulatedN production
2
(asmmolflask21).NOconcentrationsaresustainedaround transient accumulation of N2O has been studied in a
15nM (i.e. 20nmolflask21) during the period of active similarwayforcomplexcommunities[41,42],biofilms
denitrification (which lasts until all the nitrate has been [43]andincultureddenitrifiers[44–54].Itisdifficult
depleted and recovered as N ). The transient accumulation to summarize these results in a consistent way, since
2
of N O is below the detection limit of the system which is the experimental conditions are widely different from
2
approximately 2nmolflask21 . Selected data from Bergaust one experiment to the other. But the general
etal.[37]. impression is that the denitrification phenotypes of
prokaryotes are profoundly different regarding their
‘intrinsic’ propensity of emitting N O, as assessed by
2
accelerates owingtoproductstimulation (NOacceler- the amounts of N O accumulating transiently when
2
ates transcription of both genes). In contrast, nearly switching from oxic to anoxic respiration. These
100 per cent of the cells appear to express N OR, as differences between denitrification phenotypes are
2
judgedfromthespecificrateofreductionofexternally most probably owing to differences in the regulatory
supplied N O [37,38]. network controlling the transcription of their
2
ThepeculiarpatternsofregulationinP.denitrificans denitrification genes (and possibly post-translational
explains why this organism emits only traces of N O regulation). Thus, we propose a term ‘DRP’ [38],
2
(less than 2nmolflask21) during transition from oxic which is a set of variables that characterize the organ-
to anoxic conditions (figure 2). isms ability to perform a balanced and effective
The denitrification phenotype of P. denitrificans at transition from oxic to anoxic respiration with
pH 7 demonstrates an outstanding performance marginal emissions of intermediates.
regarding efficient reduction of NO all the way to Assuming that denitrifying bacteria are profoundly
x
N , with only minor emissions of both NO and N O different regarding their DRPs, a crucial question is
2 2
(less than 2nmol N O flask, or less than 0.004% of whether these characteristics are in any way related
2
allNO -NreducedtoN ).Ifthedenitrifyingcommu- to phylogeny of the organisms. If such relationships
3 2
nitiesofsoilsperformedequallywell,theircontribution exist, the analysis of denitrification community DNA
to emission of NO and N O would be negligible. The would possibly enable us to predict characteristics of
2
performance of P. denitrificans appears to be excep- the community phenotype, although the task would
tional, however. A strain that has been studied in be far from trivial owing to interactions via the inter-
equaldetailinourlaboratoryisthesoilbacteriumAgro- mediates NO2 and NO. Further on, the phylogeny
2
bacterium tumefaciens [39]. This organism is unable to of the DRP could also shed some light on the evol-
reduce N O to N because it lacks nosZ, i.e. the gene ution ofthevariousDRPtraitsandtheirfitnessvalue.
2 2
coding for N OR. Strains which lack nosZ occur However, we have scant knowledge about the phy-
2
within many genera of denitrifying prokaryotes, and if logeny of various DRP traits, since this has hardly
organisms with such truncated denitrification appar- been studied at all in the past. The results already
atus were dominating in soils, it would obviously mentioned for the closely related strains within
result in high N O/(N þN O) product ratios of deni- Thauera [40] demonstrated surprisingly different
2 2 2
trification. But A. tumefaciens contrasts with P. DRPsamongsuchcloselyrelatedstrains,bothregard-
denitrificans in another conspicuous way: when con- ingtheirtransientaccumulationofNO2andN O,but
2 2
fronted with a rapid transition from oxic to anoxic not for NO. The allocation of these DRPs within the
conditions, it appears unable to perform a balanced phylogenetic tree does not suggest any systematic
expression of NIR and NOR, resulting in extremely relationship between phylogeny and the various
highemissionsofNO,asillustratedinfigure3. DRP traits.
We have also analysed the denitrification regulatory To sum up, it seems clear that denitrification phe-
phenotype(DRP)ofanumberofreferencestrainsand notypes are profoundly different regarding their
recently isolated strains within the genus Thauera propensity to emit N O, assessed by their transient
2
under nearly identical experimental conditions as accumulation of N O when initiating denitrification
2
Phil.Trans.R.Soc.B(2012)
1230 L. R. Bakken et al. Regulation at cellular level
initial O2=˜0% initial O2=1% initial O2=7%
0.7 25 120
0.6
20 100
quid 0.5 80
M in li 00..43 1150 60
] µ20.2 40
[O 0.1 5 20
0 0 0
140 4000 12000
120
d 10000
ui100 3000
n liq 80 8000
M i 2000 6000
60
n
O] 40 4000
N 1000
[ 20 2000
0 0 0
60 60 60
50 50 50
1
–
sk 40 40 40
a
ol fl 30 30 30
m
O µ 20 20 20
2
N 10 10 10
0 20 40 60 80 100 120 140 160 0 20 40 60 80 100 120140 160 0 20 40 60 80 100 120140 160
time (h) time (h) time (h)
no added NO– 0.2 mM NO– 1 mM NO– 2 mM NO–
3 3 3 3
Figure3.DenitrificationphenotypeofAgrobacteriumtumefaciens,conditionssimilartothatforP.denitrificans(figure2),butat
threedifferentinitialconcentrationsofoxygen(indicatedattop)andfourconcentrationsofnitrate.Thetreatment‘Noadded
NO2’contains17mMNO2asapartofthebasalSistrom’smedium.AgrobacteriumtumefacienslacksN Oreductase,henceN O
3 3 2 2
istheendproduct.Atinitiallylowoxygenconcentrations((cid:2)0v/v%),thecelldensityislow,hencedepletionofoxygenisslow,
andtheorganismsareabletoperformareasonablybalancedtransitionfromoxictoanoxicdenitrification(marginalamountsof
NO accumulates transiently). At high initial oxygen concentrations (7v/v %), oxic growth secures high cell density prior to
oxygen depletion, hence final depletion of O occurs very fast, resulting in unbalanced denitrification and paralysingly high
2
NOconcetrations.ThefigureisadaptedfromLindaBergaust,PhDthesis2007,NorwegianUniversityofLifeSciences.The
figureisacorrectedversionoffig.2inBergaustetal.[39],whichhadseriouslyunderestimatedNOconcentrations(theJournal
refusedtoacceptthisasanerratum).
in response to oxygen depletion. They are also very approaches cannot be expected to reveal causal
different regarding their transient accumulation of relationshipsbetweengeneticcompositionandpheno-
nitrite (NO2). We have too few rigorous comparative type of soil microbial communities, they may serve as
2
investigations to draw conclusions on the possible hypothesis-generating observations. In order to find
relationship between these phenotypes and taxon- causal relationships, we may have to dive deeper than
omy/phylogeny, but the study of closely related to phylogeny, possibly by identifying regulatory gene
strains of Thauera do indicate that we may expect to sequencesthatcharacterizevariousDRPsacrossphyla.
find as much variation within taxonomic groups as
between them. This would mean that characterizing
the composition of denitrifying communities by meta- 5. DENITRIFICATION IS AFFECTED BY pH
genomicsequencingofgenescoding for16SrRNAor A recurring observation in ecological studies of
functional denitrification genes has limited value as a denitrificationhas been thatthe product ratioof deni-
predictor of the communities’ propensity to emit trification is strongly affected by pH [59,60]. Soils
N O.Itwouldnotimply,however,thatnorelationship have inherently different pH depending on their
2
will be found between the community composition parent mineral material modulated by biological pro-
and its propensity to emit N O. Attempts have been cesses, and the N O/(N þN O) product ratio
2 2 2 2
made, with variable success, in ecophysiological appears to be negatively correlated with pH within
studies of N O formation [55–58]. Although such the normal range for agricultural soils (pH 5–8).
2
Phil.Trans.R.Soc.B(2012)
Regulation at cellular level L. R. Bakken et al. 1231
NO– NO N O-N
2 2
µmol nmol nmol
phylogeny based on 16S rDNA
Thauera sp. DNT-1 100 19 1100
Thauera aminoaromatica 100 17 1200
Thauera phenylacetica 100 19 ---
Thauera sp. 27 <0.1 7 64
Thauera sp. 28 <0.1 13 280
Thauera linaloolentis <0.1 18 1500
Thauera terpenica 100 53 120
Thauera sp. 63 <0.1 10 5000
Azoarcus evansii
total NO : 100 µmol
0.04 0.03 0.02 0.01 0 3
Figure 4. Relationship between phylogeny and the transient accumulation of NO2, NO and N O for different strains of
2 2
Thauera;experimentalconditionsweresimilartothatusedforP.denitrificans(figure2).Thephylogenetictreewasconstructed
usingUPGMAmethod(fordetailsseeLiuetal.[40]),andthetableshowsthemaximumamountsofthethreeintermediates
transiently accumulated: NO2 (mmolflask21), NO and N O-N (nmolflask21) during batch incubations of 50ml culture
2 2
(2mM NO2) in 120ml reaction vessels. 100mmol NO2 implies that all NO2 accumulated as NO2 during the first phase
3 2 3 2
ofdenitrification.ToconvertnmolNOtonMintheliquid:1nmolflask21isequivalentto0.7nMintheliquid.ThaueraPhe-
nylaceticalackednosZ,and converted allnitrateto N O. Data assembled from Liuet al. [40].
2
This would indicate that liming slightly acidic soils Theknee-jerkreactionofmanymicrobialecologists
could be a way to reduce N O emissions, but this to this is that P. denitrificans is no more than a model
2
has been effectively ignored until recently. Why did it strain with little relevance to natural habitats. How-
take so long time to discover this? Possibly for two ever, we have collected convincing evidence that the
reasons. One is that the immediate response to phenomenon occurs in soils:
liming is a drastic increase in N O emission [61],
2
most probably because it causes a transient enhance- — Soils from long-term liming experiments demon-
ment of net N mineralization and nitrification. strate a pervasive control of pH on the (N2O/N2þ
A second reason may be that the mechanism causing N2O) product ratio, although the relative amount
the observed pH effect on the N O/N product ratio of nosZ genes and their transcripts (compared with
2 2
of denitrification was not well understood. thatoftheotherdenitrificationgenes)appearsunaf-
How can the pH affect the N O/N product ratio fected by pH within the range 5–8 [62].
2 2
of denitrification? Our studies of the model strain — Experiments with cells extracted from the same
P. denitrificans [37] shed some light on this: soils (long-term liming experiments) show essen-
tially the same phenomenon for cells from neutral
soils, but cells from acid soils appear unable to
— AtpH7,P.denitrificansemitsnearlynoN Owhen
2 produce functional N O reductase even when
2
switching from oxic to anoxic denitrification in
transferred to neutral conditions [40].
batch cultures.
— Experimentswithcellsextractedfromorganicsoils
— Lowering the pH of the medium resulted in
with different cultivation history, pH and N O
2
increasing transient accumulation of N O, and at
2 emissions [63], demonstrated the same pH
pH 6, it produces nearly 100 per cent N O (no
2 dependency, although the contrasting community
N O reductase activity).
2 composition appeared to modulate the pH
— ThelackofN OreductaseactivityatpH6wasnot
2 sensitivity [23,58].
caused by low relative transcription rate of nosZ
— All strains (Gram positives and Gram negatives)
compared with that of the other denitrification
tested in our laboratory so far have shown the
genes; the ratio between mRNA copy numbers
same pH dependency of the relative activity.
fornosZandthatfornirSwaspracticallyunaffected
— Ongoing investigationsof soils sampledfromlong-
by pH (figure 1).
term field experiments (Austria, Hungary, Nepal
— Neither isitdue toaparticularly narrow pHrange
and China) demonstrate invariably that the
fortheactivityoftheN Oreductaseenzyme(com-
2 (N O/N þN O) product ratio of denitrification
2 2 2
pared with that of the other denitrification
underanoxicincubationofintactsoilsisnegatively
enzymes): N O reductase expressed at pH 7 was
2 correlatedwithpH,independentofthemechanism
functioning well at pH 6 when tested in vivo.
controlling the soil pH (liming, parent material or
excess fertilization).
Our tentative conclusion is that low pH hinders the
synthesis of a functional N O reductase enzyme,
2
most probably by interfering with the assembly of the 6. CONCLUDING REMARKS
enzyme in the periplasm (which is the location of the Our investigations demonstrate that the N O/
2
functional enzyme). (N þN O) product ratio of denitrification is
2 2
Phil.Trans.R.Soc.B(2012)
1232 L. R. Bakken et al. Regulation at cellular level
pervasively controlled by pH, be it in pure cultures of 11 Berglund, O. & Berglund, K. 2011 Influence of water
denitrifyingbacteriaorinsoils.Ourin-depthstudiesof table level and soil properties of greenhouse gases from
the model strain P. denitrificans showed that the effect cultivated peat soil. Soil Biol. Biochem. 43, 923–931.
is primarily owing to interference with the assembly (doi:10.1016/j.soilbio.2011.01.002)
12 Dobbie, K.E. & Smith, K.A. 2006Theeffect ofwater
of the enzyme, rather than a narrow pH range for
table depth on emissions of N O from a grassland soil.
the N O reductase enzyme. The recurring obser- 2
2 Soil Use Manage. 22, 22–28. (doi:10.1111/j.1475-2743.
vations of similar pH effect on denitrification in soils
2006.00002.x)
indicate that it is a general phenomenon in nature.
13 Rochette, P. et al. 2008 Estimation of N2O emissions
Thefindingssuggestthattheongoingsoilacidification from agricultural soils in Canada. 1. Development of a
of agricultural soils by intensification of agriculture country-specific methodology. Can. J. Soil Sci. 88,
and excessive use of N-fertilizers, as convincingly 641–654.
demonstrated for China [64], may enhance the N O 14 Groffman, P. M. et al. 2006 Methods for measuring
2
emissions drastically. We hypothesize that careful denitrification:diverseapproachestoadifficultproblem.
adjustment of pH in agricultural soils will reduce Ecol. Appl. 16, 2091–2122. (doi:10.1890/1051-0761
(2006)016[2091:MFMDDA]2.0.CO;2)
N O emissions from slightly acidic soils. This needs
2 15 Kliewer,B.A.&Gilliam,J.W.1995Watertablemanage-
to be tested rigorously in field trials, however. More
ment effects on denitrification and nitrous-oxide
sophisticated ways to reduce N O emissions may
2 evolution. Soil Sci.Soc. Am.J.59, 1694–1701. (doi:10.
emerge in the future as we learn more about the ecol-
2136/sssaj1995.03615995005900060027x)
ogy and the regulatory biology of denitrifying
16 Murray, R. E. & Knowles, R. 2003 Production of NO
prokaryotes. and N O in the presence and absence of C H by soil
2 2 2
slurries and batch cultures of denitrifying bacteria. Soil
Biol. Biochem. 35, 1115–1122. (doi:10.1016/S0038-
REFERENCES 0717(03)00163-9)
1 Elser, J. J., Andersen, T., Baron,J. S., Bergstron,A. K., 17 Bergsma, T. T., Ostrom, N. E., Emmons, M. &
Jansson, M., Kyle, M., Nydick, K. R., Steger, L. & Roberstson, G. P. 2001 Measuring simultaneous fluxes
Hessen, D. O. 2009 Shifts in Lake N:P stoichiometry ofN OandN inthefieldusingthe15N-gas‘nonequili-
2 2
and nutrient limitation driven by atmospheric nitrogen brium’technique.Environ.Sci.Technol.35,4307–4312.
deposition. Science 326, 835–837. (doi:10.1126/science. (doi:10.1021/es010885u)
1176199) 18 Zaman, M., Nguyen, M. L., Matheson, F. E.,
2 Schlesinger,W.J.2009Onthefateof anthropogenicN. Blennerhassett,J.D.&Quin,B.F.2007Cansoilamend-
Proc. Natl Acad. Sci. USA 106, 203–208. (doi:10.1073/ ments(zeoliteorlime)shiftthebalancebetweennitrous
pnas.0810193105) oxideanddinitrogenemissionsfrompastureandwetland
3 Ravishankara, A. R., Daniel, J. S. & Portmann, R. W. soilsreceivingurineorurea-N?Aust.J.SoilRes.45,543–
2009 Nitrous oxide (N O): the dominant ozone- 553. (doi:10.1071/SR07034)
2
depleting substance emitted in the 21st century. Science 19 Zaman, M., Nguyen, M. L. & Saggar, S. 2008 N O
2
326, 123–125. (doi:10.1126/science.1176985) emission from pasture and wetland soils with and with-
4 Stehfest,E.&Bouwman,L.2006N OandNOemission out amendments of lime and zeolites under laboratory
2
from agricultural fields and soils under natural veg- conditions. Aust. J. Soil Sci. 46, 526–534. (doi:10.
etation: summarizing available measurement data and 1071/SR07218)
modellingofannualemissions.NutrientCycl.Agroecosyst. 20 Schurgers, G., Do¨rsch, P., Bakken, L., Leffelaar, P. &
74,207–228. (doi:10.1007/s10705-006-9000-7) Haugen, L. E. 2006 Modelling soil anaerobiosis from
5 Galloway,J.N.etal.2004Nitrogencycles:past,present water retention characteristics and soil respiration. Soil
and future. Biogeochemistry 70, 153–226. (doi:10.1007/ Biol. Biochem. 38, 2637–2644. (doi:10.1016/j.soilbio.
s10533-004-0370-0) 2006.04.016)
6 Mørkved, P. T., Do¨rsch, P. & Bakken, L. R. 2007 The 21 Chen, D., Li, Y., Grace, P. & Mosier, A. R. 2008 N2O
product ratio of nitrification and its dependence on emissions from agricultural lands: a synthesis of simu-
long term changes in pH. Soil Biol. Biochem. 39, 2048– lation approaches. Plant Soil 309, 169–189. (doi:10.
2057.(doi:10.1016/j.soilbio.2007.03.006) 1007/s11104-008-9634-0)
7 Russow, R., Stange, C. F. & Neue, H. U. 2009 Role of 22 Cavigelli,M.A.&Robertson,G.P.2000Thefunctional
nitrite and nitric oxide in the process of nitrification significanceofthedenitrifiercommunitycompositionin
anddenitrificationinsoil:resultsfrom15Ntracerexper- aterrestrial ecosystem.Ecology 81, 1402–1414. (doi:10.
iments. Soil Biol. Biochem. 41, 785–795. (doi:10.1016/j. 1890/0012-9658(2000)081)
soilbio.2009.01.017) 23 Do¨rsch,P.,Braker,G.&Bakken,L.R.2012Community
8 Bakken,L.R.&Bleken,M.A.1998Temporalaspectsof specific pH response of denitrification: experiments with
N-enrichment and emission of N2O to the atmosphere. cells extracted from organic soils. FEMS Mirobiol. Ecol.
Nutrient Cycl. Agroecosystems 52, 107–121. (doi:10. 79,530–541.(doi:10.1111/j.1574-6941.2011.01233.x)
1023/A:1009760623188) 24 Li, C. S., Frolking, S. & Butterbach-Bahl, K. 2005
9 Bakken, L. R. & Do¨rsch, P. 2007 Nitrous oxide emis- Carbon sequestration in arable soils is likely to increase
sions and global changes: modelling approaches. In nitrous oxide emissions, offsetting reductions of climate
Biology of the nitrogen cycle, Ch. 25 (eds H. Bothe, S. J. radiative forcing. Clim. Change 72, 321–338. (doi:10.
Ferguson & W. E. Newton), pp. 382–395. Amsterdam, 1007/s10584-005-6791-5)
TheNetherlands:Elsevier. 25 Frolking,S.E.etal.1998ComparisonofN2Oemissions
10 Wang,J.Y.,Jia,J.X.,Xiong,Z.Q.,Khalil,M.A.K.& from soils at three temperate agricultural sites: simu-
Xing, G. X. 2011 Water regime–nitrogen fertilizer– lations of year-round measurements by four models.
straw incorporation interaction: field study of nitrous Nutr. Cycl. Agroecosys. 52, 77–105. (doi:10.1023/
oxide emissions from a rice agroecosystem in Nanjing, A:1009780109748)
China. Agric. Ecocyst. Environ. 141, 437–446. (doi:10. 26 Hillier, J., Walter, C., Malin, D., Garcia-Suarez, T.,
1016/j.agee.2011.04.009) Mila-i-Canals, L. & Smith, P. 2001 A farm-focused
Phil.Trans.R.Soc.B(2012)
Regulation at cellular level L. R. Bakken et al. 1233
calculator for emission from crop and livestock pro- directionality of autotrophic nitrous oxide and nitric
duction. Environ. Model. Softw. 26, 1070–1078. oxide generation during transient anoxia. Environ. Sci.
(doi:10.1016/j.envsoft.2011.03.014) Technol.44,1313–1319.(doi:10.1021/es902794a)
27 Insam,H.&Wett,B.2008ControlofGHGemissionat 43 Schreiber, F., Loeffler, B., Polerecky, L., Kuypers,
the microbial community level. Waste Manage. 28, M.M.M.&deBeer,D.2009Mechanismsoftransient
699–706. (doi:10.1016/j.wasman.2007.09.036) nitric oxide and nitrous oxide production in a complex
28 Luo,J.,deKlein,C.A.M.,Ledgard,S.F.&Saggar,S. biofilm. ISME J. 3, 1301–1313. (doi:10.1038/ismej.
2010Managementoptionstoreducenitrousoxideemis- 2009.55)
sions from intensively grazed pastures: a review. Agric. 44 Kester,R.A.,deBoer,W.&Laanbroek,H.J.1997Pro-
Ecosyst. Environ. 136, 282–291. (doi:10.1016/j.agee. duction of NO and N O by pure cultures of nitrifying
2
2009.12.003) and denitrifying bacteria during changes in aereation.
29 Liebig,M.A.,Morgan,J.A.,Reeder,J.A.,Ellert,B.H., Appl. Environ. Microbiol. 63,3872–3877.
Gollany,H.T.&Schuman,G.E.2005Greenhousegas 45 Miyahara,M.etal.2010Potentialofaerobicdenitrifica-
contributions and mitigation potential of agricultural tionbyPseudomonasstutzeriTR2toreducenitrousoxide
practices in northwestern USA and Western Canada. emissions from waste water treatment plants. Appl.
Soil Tillage Res. 83, 25–52. (doi:10.1016/j.still.2005.02. Environ. Microbiol. 76, 4619–4625. (doi:10.1128/AEM.
008) 01983-09)
30 Johnson, J. M. F., Reicosky, D. C., Allmaras, R. R., 46 Baumann,B.,Snozzi,M.,VanderMeer,J.R.&Zehnder,
Sauer, T.J., Venterea, R. T. & Dell, C. J. 2005 Green- A.J.B.1997Developmentofstabledenitrifyingcultures
house gas contributions and mitigation potential of during repeated aerobic–anaerobic transient periods.
agriculture in the central USA. Soil Tillage Res. 83, Water Res. 31, 1947–1954. (doi:10.1016/S0043-
73–94. (doi:10.1016/j.still.2005.02.010) 1354(97)00053-5)
31 Albrecht,A.&Kandij,S.T.2003Carbonsequestration 47 Mahne, I. & Tiedje, J. M. 1995 Criteria and method-
in tropical agroforestry systems. Agric. Ecosyst. Environ. ology for identifying respiratory denitrifiers. Appl.
99, 15–27.(doi:10.1016/S0167-8809(03)00138-5) Environ.Microbiol.61,1110–1115.
32 Granli, T. & Bøckman, O. C. 1994 Nitrous oxide from 48 Otte, S., Grobben, N. G., Robertson, L. A., Jetten,
agriculture.Norwegian J.Agric. Sci.Suppl. 12,1–128. M. S. M. & Kuenen, J. G. 1996 Nitrous oxide pro-
33 Van Spanning, R. J. M., Richardson, D. & Ferguson, S. duction by Alcaligenes faecalis under transient and
2007 Introduction to the biochemistry and molecular dynamic aerobic and anaerobic conditions. Appl.
biology of denitrification. In Biology of the nitrogen cycle, Environ.Microbiol.62,2421–2426.
Ch. 1 (eds H. Bothe, S. J. Ferguson & W. E. Newton), 49 McKenney,D.J.,Drury,C.F.,Findlay,W.I.,Mutus,B.,
pp.382–395.Amsterdam,TheNetherlands:Elsevier. McDonnell,T.&Gajda,C.1994Kineticsofdenitrifica-
34 Zumft, W. 1997 Cell biology and molecular tion by pseudomonas fluorescence—oxygen effects. Soil
basis of denitrification. Microbiol. Mol. Biol. Rev. 61, Biol. Biochem. 26, 901–908. (doi:10.1016/0038-
533–616. 0717(94)90306-9)
35 Zumft,W.&Kroneck,P.M.H.2007Respiratorytransform- 50 Kornaros,M.&Lyberatos,G.1998Kineticmodellingof
ationofnitrousoxide(N2O)todinitrogenbyBacteriaand Pseudomonas denitrificans growth and denitrification
Archaea.Adv.Microbial.Physiol.52,109–197. underaerobic,anoxicandtransientoperatingconditions.
36 Bergaust,L.,vanSpanning,R.,Frostega˚rd,A˚.&Bakken, Water Res. 32, 1912–1922. (doi:10.1016/S0043-
L. R. In press. Expression of nitrous oxide reductase in 1354(97)00403-X)
Paracoccus denitrificans is regulated by oxygen and nitric 51 Remde, A. & Conrad, R. 1991 Metabolism of nitric
oxide through FnrP and NNR. Microbiology (doi:10. oxide in soil and denitrifying bacteria. FEMS Microbiol.
1099/mic.0.054148-0) Ecol. 85, 81–93. (doi:10.1111/j.1574-6968.1991.
37 Bergaust, L., Mao, Y., Bakken, L. R. & Frostega˚rd, A˚. tb04700.x)
2010 Denitrification response patterns during the tran- 52 Cheneby,D.,Perrez,S.,Devroe,C.,Hallet,S.,Couton,
sition to anoxic aespiration and posttranscriptional Y.,Bizouard,F.,Iuretig,G.,Germon,J.C.&Philippot,
effects of suboptimal pH on nitrogen oxidereductase in L. 2004 Denitrifying bacteria in bulk and maize-
Paracoccus denitrificans. Appl. Environ. Microbiol. 76, rhizospheric soil: diversity and N2O reducing abilities.
6387–6396. (doi:10.1128/AEM.00608-10) Can. J. Microbiol. 50,469–474.
38 Bergaust,L.,Bakken,L.R.&Frostegard,A.2011Deni- 53 Conrad, R. 1996 Soil microorganisms as controllers of
trification regulatory phenotype, a new term for the atmospheric trace gases (H , CO, CH , OCS, N O
2 4 2
characterization of denitrifying bacteria. Biochem. Soc and NO). Microbiol. Rev. 60,609–640.
Trans.39,207–212.(doi:10.1042/BST0390207) 54 Kanako,T.,Ishii,S.,Nishizawa,T.,Otsuka,S.&Senoo,
39 Bergaust, L., Shapleigh, J., Frostega˚rd, A˚. & Bakken, K.2011Phylogeneticandfunctionaldiversityofdenitri-
L.R.2008TranscriptionandactivitiesofNOxreductases fying bacteria isolated from various Rice Paddy and
in Agrobacterium tumefaciens: the influence of nitrite, Rice-Soybean Rotating fields. Microbes Environ. 26,
nitrate and oxygen availability. Environ. Microbiol. 10, 30–35.(doi:10.1264/jsme2.ME10167)
3070–3081.(doi:10.1111/j.1462-2920.2007.01557.x) 55 Ma, W. K., Bedard-Haughn, A., Siciliano, S. D. &
40 Liu,B.,Mao,Y.,Bakken,L.R.&Frostega˚rd,A˚.Inprep- Farrel, R. E. 2008 Relationship between nitrifier and
aration. A comparative analysis of genotypes and denitrifier community composition and abundance in
denitrification phenotypes of strains within the genus predicting nitrous oxide composition from ephemeral
Thauera. wetland soils. Soil Biol. Biochem. 40, 1114–1123.
41 Mao,Y.,Bakken,L.R.,Zhao,L.&Frostega˚rd,A.2008 (doi:10.1016/j.soilbio.2007.12.004)
Functional robustness and gene pools of a wastewater 56 Balser, T. C. & Firestone, M. F. K. 2005 Linking
nitrification reactor: comparison of dispersed and intact microbial community composition and soil processes in
biofilms when stressed by low oxygen and low pH. a California annual grassland and mixed-conifer forest.
FEMS Microbiol. Ecol. 66, 167–180. (doi:10.1111/j. Biogeochemistry 73, 395–415. (doi:10.1007/s10533-004-
1574-6941.2008.00532.x) 0372-y)
42 Yu,R.,Kampschreur,M.J.,vanLoosdrecht,M.C.M.& 57 Rich, J. J. & Myrold, D. D. 2004 Community
Chandran, K. 2010 Mechanisms and specific composition and activities of denitrifying bacteria from
Phil.Trans.R.Soc.B(2012)
1234 L. R. Bakken et al. Regulation at cellular level
adjacent agricultural soil, riparian soil and creek 61 Baggs, E. M., Smales, C. M. & Bateman, E. J. 2010
sediment in Oregon, USA. Soil. Biol. Biochem. 36, Changing pH shifts the microbial source as well as
1431–1441. (doi:10.1016/j.soilbio.2004.03.008) the magnitude of N2O emission. Biol. Fertil. Soils 46,
58 Braker, G., Do¨rsch, P. & Bakken, L. R. 2012 793–805. (doi:10.1007/s00374-010-0484-6)
Genetic characterizationof denitrifiercommunitieswith 62 Liu,B.,Mørkved,P.T.,Frostega˚rd,A˚.&Bakken,L.R.
contrasting intrinsic functional traits. FEMS Mirobiol. 2010 Denitrification gene pools, transcription and kin-
Ecol. 79, 542–554. (doi:10.1111/j.1574-6941.2011. etics of NO, N O and N production as affected bysoil
2 2
01237.x) pH. FEMS Microbiol. Ecol. 72, 407–417. (doi:10.1111/
59 Firestone,M.K.,Firestone,R.B.& Tiedje,J.M.1980 j.1574-6941.2010.00856.x)
Nitrous oxide from soil denitrification: factors control- 63 Holtan-Hartwik, L., Do¨rsch, P. & Bakken, L. R. 2000
ling its biological production. Science 208, 749–751. Comparison of denitrifying communities in organic
(doi:10.1126/science.208.4445.749) soils: kinetics of NO2 and N O reduction. Soil Biol.
3 2
60 Simek, M. & Cooper, J. E. 2002 The influence of soil Biochem. 32, 833–843. (doi:10.1016/S0038-0717(99)
pH on denitrification: progress towards the under- 00213-8)
standing of this interaction over the last 50 years. 64 Guo, J. H. et al. 2010 Significant acidification in major
Eur. J. Soil Sci. 53, 345–354. (doi:10.1046/j.1365- Chinese croplands. Science 327, 1008–1010. (doi:10.
2389.2002.00461.x) 1126/science.1182570)
Phil.Trans.R.Soc.B(2012)