Final Revised Paper - Biogeosciences

13 Pages·2013·0.84 MB·English

EGU Journal Logos (RGB)
O O O
p p p
Advances in e Annales e Nonlinear Processes e
n n n
A A A
Geosciencesc Geophysicaec in Geophysicsc
c c c
e e e
s s s
s s s
Natural Hazards O Natural Hazards O
p p
e e
and Earth System n A and Earth System n A
c c
c c
Sciencese Sciencese
s s
s s
Discussions
Atmospheric O Atmospheric O
p p
e e
n n
Chemistry A Chemistry A
c c
c c
and Physicse and Physicse
s s
s s
Discussions
Atmospheric O Atmospheric O
p p
e e
n n
Measurement A Measurement A
c c
c c
Techniquese Techniquese
s s
s s
Discussions
Biogeosciences,10,1155–1167,2013 O O
p p
www.biogeosciences.net/10/1155/2013/ e Biogeosciencese
n n
doi:10.5194/bg-10-1155-2013 Biogeosciences A A
c c
c Discussions c
©Author(s)2013. CCAttribution3.0License. e e
s s
s s
O O
p Climate p
Climate e e
n n
A of the Past A
of the Pastc c
c c
e e
s Discussionss
s s
An unknown oxidative metabolism substantially contributes to soil
O O
CO emissions p Earth System p
2 Earth System e e
n n
A Dynamics A
Dynamicsc c
V.Maire1,*,G.Alvarez1,2,J.Colombet3,A.Comby1,R.Despinasse4,E.Dubreucq5,M.Joly1,A.-C.Lehoucers3, ce
s Discussionss
V.Perrier5,T.Shahzad1,**,andS.Fontaine1 s s
1INRA,UR874UREP,63100Clermont-Ferrand,France
Geoscientific Geoscientific
2ClermontUniversite´,VetAgroSup,63000,Clermont-Ferrand,France O O
p p
3UniversityofClermontFerrand,UMR6023LMGE,63177Aubie`re,France Instrumentation en Instrumentation en
4INRA,UMR1095UBP,63100Clermont-Ferrand,France Methods and Ac Methods and Ac
c c
5MontpellierSupAgro,UMR1208IATE,34060Montpellier,France Data Systemsess Data Systemsess
*presentaddress:DepartmentofBiologicalSciences,MacquarieUniversity,NSW2109,Australia
**presentaddress:DepartmentofEnvironmentalSciences,GovernmentCollegeUniversityFaisalabad, Discussions
38000-AllamaIqbalRoadFaisalabad,Pakistan O GeoscientificO
p p
Geoscientifice e
n n
Correspondenceto:S.Fontaine([email protected]) A Model Development A
Model Developmentcc cc
e e
s Discussionss
Received:31May2012–PublishedinBiogeosciencesDiscuss.:18July2012 s s
Revised:25January2013–Accepted:27January2013–Published:21February2013
Hydrology and O Hydrology and O
p p
e e
Ajobrsdtertaecrtm.iTnhaentreosfptihraetogrloybraellecaasreboonfCcyOc2lef.roItmisstorialdsiitsioanmallay- lgylsutcso.sFeorreiqnustiarenscea,ttehmeppehEryastaiucrraetlhgoSrx eciSadtiaeeytrisnothntceaonefm5so0n0n Accee◦mCo.lIencusloeilosf, EarthS cSieysntceemsn Acce
s s
consideredthatthisrespirationisanintracellularmetabolism microorganismssetupacomplexcascadeofbisochemicalre- s
consisting of complex biochemical reactions carried out by actionsmediatedbynumerousenzymesmakingtheoxidisa- Discussions
numerous enzymes and co-factors. Here we show that the tionoforganicCpossibleatlowtemperature. O O
p p
endoenzymesreleasedfromdeadorganismsarestabilisedin The mineralisation of organic C requires tweo main steps. Ocean Sciencee
n n
soilsandhaveaccesstosuitablesubstratesandco-factorsto First, microorganismsOseccreetaenex tSracceilelunlacreenz Aymes in soil A
cc Discussions cc
permitfunction.Theseenzymesreconstituteanextracellular in order to deconstruct plant and microbial ceell walls (e.g., e
s s
oxidativemetabolism(EXOMET)thatmaysubstantiallycon- endoglucanase),depolymerizemacromoleculess(e.g.,perox- s
tribute to soil respiration (16 to 48% of CO released from ydase),andultimatelyproducesolublesubstratesformicro-
2
soilsinthepresentstudy).EXOMETandrespirationfromliv- bialassimilation(e.g.,glucosidase)(Chro´st,199O1;Burnsand O
p p
ing organisms should be considered separately when study- Dick,2002;Sinsabaughetal.,2009).NomolecenuleofCO2is Solid Earthen
ing effects of environmental factors on the C cycle because releasedduringthisdepolymSeriozalitdio nEstaepr.thTh Accesecondstep Discussions Acc
EXOMETshowsspecificpropertiessuchasresistancetohigh ofmineralisation,duringwhichCisreleasedasesCO2,implies es
s s
temperatureandtoxiccompounds. theabsorptionandutilisationofsolubilizedsubstratesbymi-
crobialcellswiththeaimtoproduceenergy(ATP).Incells,
soluble substrates are carried out by a cascadOe of endoen- O
p p
zymes (enzymes contained by membranes ofenliving cells; The Cryosphereen
1 Introduction Sinsabaughetal.,20T12h),eal oCngrywohischpphreotroens Acandelectrons Ac
c Discussions c
e e
are transferred from a substrate to an approprsiate acceptor s
s s
Knowledge of the metabolic pathways through which or- (e.g.,theaerobicrespirationaswellastheanaerobicrespira-
ganic carbon (C) is oxidised into carbon dioxide (CO2) in tionandthefermentationwithdifferentfinalelectronaccep-
soilsisfundamentaltounderstandingtheglobalCcycleand tors;Prescottetal.,2002).Underaerobicconditions,theox-
itsinteractionswithclimate(Fontaineetal.,2007;Heimann idativemetabolismleadstoproductionofadenosinetriphos-
andReichstein,2008).OrganicCisthebuildingblockofliv- phate (ATP), consumption of oxygen (O ) and emission of
2
ingorganismsandisdefactohighlystableinabsenceofcata-
PublishedbyCopernicusPublicationsonbehalfoftheEuropeanGeosciencesUnion.
1156 V.Maireetal.: AnunknownoxidativemetabolismcontributestosoilCO emissions
2
CO2. Respiration fluxes and ATP production in soils are zymeswithglucoseinsterilisedwaterandsoil.TheEXOMET
usually positively correlated with activity of dehydrogenase induced by the yeast-extract was quantified by measuring
endoenzymes (e.g., Casida et al., 1964), but only some of CO and O fluxes in water and soil microcosms. Second,
2 2
themcarryadecarboxylasefunction(e.g.,pyruvatedehydro- the protective role of soil particles (minerals and humus)
genase, oxoglutarate dehydrogenase) that cut the carboxyl for respiratory enzymes was tested by incubating three en-
functionoforganicCmoleculestoliberateCO . zymesinvolvedinglycolysisandtheKrebscycleinfivetop
2
The function of respiration-carrying endoenzymes de- soils sampled from different regions of the world (Table 1).
pends on various co-factors that must be regenerated (e.g., Thethirdpartofthemanuscriptisdevotedtothequantifica-
NAD+), on being located adjacent to other enzymes and tionofEXOMETcontributiontotheCO2emissionsfromfive
on physiological properties of the cell (e.g., redox poten- studiedsoils.Thiscontributionwasquantifiedwithamethod
tial) (Krebs, 1981; Rich, 2003). Given this complexity and combiningmodellingandincubationsofsterilisedandnon-
the typical fragility of respiratory enzymes, it is tradition- sterilisedsoils.Finally,westudiedsomeEXOMETproperties
ally considered that respiration (second step of C minerali- by incubating soils exposed to high temperature, high pres-
sationprocess)isstrictlyanintracellularmetabolismprocess sureandtoxiccompounds.
(Burns, 1982; Makoi and Ndakidemi, 2008). Besides, from
an evolutionary point of view, soil microorganisms have no
2 Materialsandmethods
interest in provoking extracellular respiration since the en-
ergy resulting from this respiration (ATP) may escape from
2.1 Soilsamplingandsterilisation
the cell that provided enzymes (Allison et al., 2011). How-
ever, some studies have shown that substantial emission of
Foreachofthefivestudiedsites(Table1),twentyindepen-
CO can persist for several weeks in soils where microbial
2 dentsoilsampleswerecollectedfromthe0–20cmsoillayer.
life has been reduced by exposition to toxics (ClCH , or-
3 Soilsamplesfromeachsitewerepooledtomakeacomposite
angeacridine)orirradiationtoundetectablevalue(Peterson,
samplepersite.Soilwassievedat2mmandwasthenused
1962;RamsayandBawden,1983;Lensietal.,1991;Trevors,
to determine pH, texture and organic matter content and to
1996; Kemmitt et al., 2008). Such emission of CO could
2 conductincubationexperiments.Thefivesoilspresentedtex-
not be explained by an activity of surviving microbes that
tures from sandy-silted to silty-clay soils, pH ranging from
wouldbepresentinlowquantityinsoils,unlessconsidering
4.3 to 8.6 and three types of land use (grassland, forest and
unrealisticallyhighcellrespiratoryactivity.Neithercouldit
crops,Table1).ThesoilfromTheixwasusedforallinves-
be explained by the activity of extracellular enzymes previ-
tigations,whereastheothersoilswereusedtogeneralisethe
ously secreted by soil microorganisms since these enzymes
keyfindingsofthisstudy.
only solubilize organic C and do not release CO . To date,
2 Someincubationexperimentsinvolvedtheuseofsterilised
thecauseofCO emissioninsoilswheremicrobiallifehas
2 soils. We tested different methods of soil sterilisation (γ-
beenminimisedseemstobeunknownandquestionsourba-
irradiation,autoclavinganddryheating)duringpreliminary
sicknowledgeofbiology.
investigations. Irradiation was chosen among other sterilis-
The objective of this study is to understand the cause
ing methods for its efficiency to kill soil micro-organisms
of CO emission in soils where microbial life has been
2 and for its moderate effect on soil enzymes (see S1 for de-
suppressed. We hypothesise that an extracellular oxidative
tailsofinvestigationsonsoilsterilisation).Thepreservation
metabolism (EXOMET) can be reconstituted by respiratory
ofsoilenzymeswasimportantforquantifyingthe EXOMET
endoenzymesreleasedfromdeadorganismsinsoilsandthat
contributiontosoilCO emissions(seetheSect.2.4).Soils
2
this EXOMET cansubstantiallycontributetosoilCO2 emis- were sterilised by γ-irradiation at 45kGy (60Co, IONISOS,
sion.Themanuscriptisorganisedaccordingtofourkeyques-
ISO14001,France).
tions:
1. Can an oxidative metabolism occur in an extracellular 2.2 DemonstrationofEXOMETbyincubatingacell-free
yeast-extractinsterilisedwaterandsoil
context?
2. How long can this EXOMET persist within soil? What 2.2.1 Productionofcell-freeyeast-extract
is the role of soil particles for EXOMET-carrying en-
zymes? PichiapastorisX33(Invitrogen,Clareetal.,1991)cellswere
culturedat28◦Cina1.5LcapacityApplikonbioreactorcon-
3. How much does EXOMET contribute to CO2 emission taining synthetic medium described by Boze et al. (2001).
fromlivingsoil? This medium contained 80 µgL−1 D-biotin, 40gL−1 glyc-
erolandmineralsolutions(FM21andPTM1).ThepHofthe
4. WhatarethespecificpropertiesofEXOMET?
mediumwasregulatedat5usingaNH OHsolution(15%,
4
First, to assess the possibility of an EXOMET, we in- v/v)thatalsoservedasanitrogensource.DissolvedO2was
cubated a cell-free yeast-extract containing respiratory en- measuredusingapolarographicprobeandwasmaintainedat
Biogeosciences,10,1155–1167,2013 www.biogeosciences.net/10/1155/2013/
V.Maireetal.: AnunknownoxidativemetabolismcontributestosoilCO emissions 1157
2
Table1.Sitecharacteristicsandsoilproperties.
Sites
Bugac Laqueuille PontaGrossa Sorø Theix
Country Hungary France Brazil Denmark France
Landuse Grassland Grassland Crops Beechforest Grassland
Clay/Silt/Sand(%) 10/5/85 19/55/26 14/6/80 10/22/68 26/25/49
pH 8.6 5.3 7.0 4.3 6.2
OMcontent(gCkg−1) 44.0 121.9 14.8 33.7 39.0
CEC(cmol+kg−1) 10.2 25.1 3.9 9.5 21.5
over30%saturationbystirringupto1800rpmandaeration sterilised by filtration at 0.2 µm. Three replicates per treat-
(an injection of 1L of air per litre of medium per minute). ment were prepared. Soils were incubated at a water poten-
Dataacquisitionandbioprocesscontrolwerecarriedoutus- tialof−100kPa.Waterandsoilmicrocosmswereincubated
ingtheApplikonbiocontrollersoftware.Biomassconcentra- at30◦Cfor53days.The EXOMET inducedbythecell-free
tionofculturesampleswasdeterminedbyweighingwashed yeastextractwasquantifiedbymeasuringtheconcentration
cellsafterdryingat105◦Ctoconstantweight. ofCO ,13CO andO inwaterandsoilmicrocosmsthrough-
2 2 2
The yeast culture was harvested at the end of the outthe53daysofincubation(seeSect.2.6fordetailsonflux
growth phase by centrifugation at 25000xg for 10min measurements). All manipulations were done under sterile
at 5◦C to yield a hard pellet. To remove the culture conditions and the sterility of microcosms was verified af-
media, the sediment was washed and re-decanted three ter13and53daysofincubationthroughdirectfluorescence
timesinpotassiumphosphatebuffer(0.1M,pH=6.5which microscopic observations and TSA-FISH method (see Sup-
corresponded to the pH of the Theix soil used for the plementS1).
experiment, V /V =1/10). The sediment was re-
yeast buffer
suspendedinasmallvolumeofpotassiumphosphatebuffer 2.3 Soilstabilisationofoxidativemetabolismenzymes
(V /V =2/1) before cells were disrupted with a
yeast buffer
French pressure cell press (100 MPa, Vanderheiden et al., 2.3.1 Enzymeincubation
1970). Unbroken cells and large cell debris were removed
fromtheyeastextractbyfourteensuccessivecentrifugations
In order to quantify the protective role of soil par-
at 25000xg and 5◦C. The yeast extract was filtered under
ticles (minerals, humus) for respiratory enzymes, three
sterileconditionsat0.2 µmtoobtainacell-freeextract.Di-
microbial enzymes involved in glycolysis (GHK: glu-
rectmicroscopicobservationconfirmedsterilityoftheyeast
cose hexokinase EC.2.7.1.1, Sigma-Aldrich ref H4502;
extract(S1).Thecell-freeyeastextractwasimmediatelyin-
G6PI: glucose-6-phosphate isomerase EC.5.3.1.9, Sigma-
corporatedintowaterandsoilmicrocosmsundersterilecon-
Aldrich ref P5381) and the Krebs cycle (MDH: malate de-
ditions.Thecell-freeyeastextractcontained28.5mgprotein
hydrogenase, EC.1.1.1.37, Sigma-Aldrich ref M7032) were
(Biuret method, Okutucu et al., 2007) and 0.77unitL−1 of
separately incubated in the non-irradiated-soil from Theix.
malatedehygrogenase(MDH).
A solution with an enzymatic activity of approximately
50UL−1 waspreparedforeachenzyme.Avolumeof15µL
2.2.2 Yeastextractincubationinsterilisedwater ofenzymesolutionwasincorporatedin80mgsoil(drymass
andsoil basis) and incubated at 20◦C and −100kPa. Soils without
enzyme amendment were also incubated as controls. Two
The cell-free yeast extract (YE) was incubated in sterilised sets of each treatment (controls without enzyme and soils
water(W)orintheirradiated-soil(S)ofTheix(Table1).Ex- with enzyme) were prepared in order to separately quantify
perimental microcosms consisted of 5mL of cell-free yeast the activity of total, soluble and soil-immobilised enzymes.
extractand1mLof13Clabelledglucosewithorwithout20g Immobilisedenzymeswereassumedtobeprotectedfromde-
ofγ-irradiated-soil(S+G+YEandW+G+YEtreatments, naturationandproteolysis(Burns,1982;Sarkaretal.,1989;
respectively)placedin250mLairtightflasks.Aseconddose Quiquampoix,2000)andexpectedtomaintaintheiractivity
ofglucosewasappliedaftertwentydaysofincubationinor- inthelong-term(>tendays).Differentindependentsubsets
dertodeterminethepersistenceoftheEXOMET.Waterwith ofsoilsampleswerepreparedtomeasuretheactivityofen-
glucoseandirradiated-soilwithorwithout13Clabelledglu- zymesatdifferenttimesbetween20minand35daysofen-
cosewereincubatedascontrolsamples(W+G,SandS+G zyme incubation in soils. The protective role of soil on res-
treatments, respectively). The 13C labelled glucose solution piratoryenzymeswasgeneralisedagainstthefourothersoils
(δ13C=3712‰)contained60mgC-glucosemL−1andwas varyingintextureandorganicmattercontentbyrepeatingthe
www.biogeosciences.net/10/1155/2013/ Biogeosciences,10,1155–1167,2013
1158 V.Maireetal.: AnunknownoxidativemetabolismcontributestosoilCO emissions
2
A
. Glucose hexokinase (GHK E.C. 2.7.1.1)
Glucose PENTOSE PHOSPHATE
ATP Glucose PATHWAY

hexokinase
ADP NADP+ NADPH + H+ H2O
GLYCOLYSIS
Glucose-6- Mg2+ Phosphoglucono- Mg2+ 6-phospho
phosphate G6P δ-lactone Lactonase gluconate
dehydrogenase
G6P
isomerase
Fructose-6-
phosphate
B
. Glucose-6-phosphate isomerase (G6PI EC 5.3.1.9)
Glucose PENTOSE PHOSPHATE
ATP Glucose PATHWAY
hexokinase
ADP NADP+ NADPH + H+ H2O
GLYCOLYSIS
Glucose-6- Mg2+ Phosphoglucono- Mg2+ 6-phospho
phosphate G6P δ-lactone Lactonase gluconate
dehydrogenase
G6P
isomerase
Fructose-6-
phosphate
C
. Malate dehydrogenase (MDH EC 1.1.1.37)
CoA-SH Pyruvate dehydrogenase
NAD Dihydrolipoyl transacetylase
Dihydrolipoate dehydrogenase
NADH + H+
CO2
Acetyl-CoA
NAD+ NADH + H+ CoA-SH
Mg2+
L-Malate Oxaloacetate Citrate
Malate Citrate synthase
dehydrogenase
KREBS CYCLE

Fig. 1. Enzymatic rea ctions mediated by the two enzymes involved in glycolysis ((A) GHK: glucose hexokinase, (B) G6PI: glucose-6-
phosphateisomerase)andtheenzymeinvolvedintheKrebscycle((C)MDH:malatedehydrogenase)usedtoquantifytheprotectiveroleof
soilparticlesonrespirFaitgouryreen 1z ymes.Thisprotectiverolewasstudiedinthenon-irradiated-soilfromTheix.Theprincipleoftheenzymatic
activitymeasurementwastosetupasystemofenzymaticreactionswherethestudiedenzyme(red)wasthelimitingfactor.Fordoingthis,
weaddedallsubstrates,cofactorsandintermediaryenzymesinexcess(green).Theactivityofthestudiedenzyme(GHK,G6PI,MDH)was
quantifiedbymeasuringtheformationortheconsumptionofNADHbyspectrometry.Reactionssurroundingthestudiedenzymatic(grey)
aregivenforinformation.
aboveexperimentandbyusingtheG6PIasamodelenzyme al., 2007). To this end, substrates, cofactors and intermedi-
(seeSupplementS2). ary enzymes of the enzymatic reaction system were mixed
in a buffer solution and incorporated in excess in soil sam-
2.3.2 Principleofenzymeactivitymeasurement ples (green in Fig. 1). The activity of each studied enzyme
(GHK,G6PI,MDH)wasquantifiedbymeasuringtheforma-
The principle of the enzymatic activity measurement was
tionortheconsumptionofNADHbyspectrometryfollowing
to set up a system of enzymatic reactions where the stud-
Nardietal.(2007),withsomeprotocolmodificationsforsoil
ied enzyme (red in Fig. 1) was the limiting factor (Nardi et
Biogeosciences,10,1155–1167,2013 www.biogeosciences.net/10/1155/2013/
V.Maireetal.: AnunknownoxidativemetabolismcontributestosoilCO emissions 1159
2
conditionsasopposedtoleaves.Inparticular,concentrations ment of total enzyme activity (TotalEnz), the enzymatic re-
ofbufferandMg++solutionwereincreasedinordertobetter actionwasmadeintothesoilinpresenceofsolubleandsoil-
controlsoilpHandprecipitatehumicacidsthatcouldhamper immobilisedenzymes.Tothisend,soil-enzymemixturewas
the quantification of NADPH by spectrometry (both humic incubatedwithsubstratesandcofactors(seeSect.2.3.2)dur-
acids and NADPH absorb at 340nm). For each of the three ing45min.Atdifferenttimesbetween5and45minofincu-
enzymes, the enzymatic reaction system and the method of bation with substrates, independent samples were harvested
enzymeactivitymeasurementwereasfollows: and centrifuged at 11000xg during 3min. The NADPH
concentration in the supernatant was determined by spec-
– GHK(Fig.1a):SoilwithorwithoutGHKwasamended
trophotometry at 340nm. The production of NADPH (for
with 300µL of solution containing a buffer (Bicine-
GHK and G6PI) or the consumption of NADH (for MDH)
NaOH,100mM,pH=8.5),glucose(1.5mM),NADP+
during the 45min incubation of soil with substrates corre-
(1.5mM),Mg++(32mM)andglucose-6-phosphatede-
spondedtotheactivityoftotalenzymes.
hydrogenase (EC‘1.1.1.49, Sigma-Aldrich ref G5885).
Production of NADPH following the chemical trans- 2.3.4 Kineticanalysisoftotalenzymeactivity
formationofglucoseinphosphoglucono-δ-lactonewas
measuredbyspectrometryat340nm. Different exponential models were tested to fit the decrease
in total enzyme activity (Total Enz) with time. The sole ex-
– G6PI(Fig.1b):SoilwithorwithoutG6PIwasamended ponential model able to explain kinetics of total enzymatic
with 300µL of solution containing a buffer (Bicine- activityconsidersthreepoolsofenzymes:
NaOH, 100mM, pH=8.5), fructose-6-phosphate
(1.5mM), NADP+ (1.5mM), Mg++ (32mM) and Y(t)=a·exp(−b·t)+c·exp(−d·t)+f ·exp(−g·t) (1)
glucose-6-phosphate dehydrogenase (2.5UL−1).
ProductionofNADPHfollowingthechemicaltransfor- where t is the time of incubation, a, c and f represent the
mation of fructose-6-phosphate to phosphoglucono-δ- sizesandb,d andgthedecayratesoffast,intermediateand
lactonewasmeasuredbyspectrometryat340nm. slow pools, respectively. Decay rate of the fast pool was so
rapidthatitcouldnotbecharacterisedpreciselyinthisexper-
– MDH (Fig. 1c): Soil with or without MDH was iment.Consequently,sizeaanddecayratebofthefastpool
amended with 300µL of solution containing a buffer were fixed to the amount of enzymatic activity lost during
(TRIS 100mM, pH=6.7), oxaloacetate (1.5mM), thefirst20minofincubationandto1/5min−1.Then,param-
NADH(1.5mM)andMg++ (32mM).Consumptionof etersc,d,f andgofintermediateandslowpoolsofenzymes
NADH following the chemical transformation of ox- couldbeestimatedusingaclassicalnonlinearregressionpro-
aloacetate to L-malate was measured by spectrometry cedure.Weverifiedthatuncertaintyondecayrateofthefast
at340nm. poolhasnegligibleeffectonestimationofparametersofthe
intermediate and slow pools. This lack of effect on the es-
2.3.3 Activityoftotal,solubleandsoil-immobilised timationofparametersisexplainedbythestrongdifference
enzymes betweendecayrateofthefastpoolandthoseofintermediate
and slow enzyme pools (data not shown). The half-lives of
For each endoenzyme, activity of total, soluble and soil-
theenzymepoolswerecalculatedas(ln2)/(decayrate).
immobilised enzymes was estimated at different times be-
tween20minand35daysofenzymeincubationinsoils.At 2.4 ContributionofEXOMETtosoilrespiration
each harvest date, two independent sets of soils were used
toquantifyactivityoftotal(TotalEnz)andsolubleenzymes AmethodbasedonthemeasurementofCO emissionsfrom
2
(SoluEnz). Activity of soil-immobilised enzymes was esti- irradiated and non-irradiated soils (see the Sect. 2.4.2) was
mated by difference (ImmEnz=TotalEnz−SoluEnz). Ac- developed to quantify EXOMET and the respiration of liv-
tivity of soluble enzyme (SolEnz) was quantified after their ing organisms in the five studied soils. Gamma irradiation,
extractionfromsoil.Forextraction,80mgsoilsampleswere by killing the soil organisms, was considered to stop living
mixedwiththe300µlofthebuffersolutioncontainingsub- respiration whilst preserving soil enzymes (S1) responsible
strates,co-factorsandintermediateenzymes(seeSect.2.3.2) forEXOMET.Therefore,CO2emissionsfromirradiatedsoils
and shaken during 5min. Then, samples were centrifuged wereusedtoestimatethe EXOMET.However,γ-irradiation
at 11000xg during 3min. The supernatant containing sol- alsoreleasedalargequantityofendoenzymesfromthekilled
ubleenzymes,co-factorsandsubstrateswastransferredinto organisms,therebyartificiallyincreasingtheEXOMETinthe
amicro-plate,whereactivityofsolubleenzymeactivitywas irradiated-soils.Takingthiseffectintoaccount,amodelofC
measured during 3min. The production rate of NADPH fluxwasusedtoquantifyEXOMETandlivingrespiration.
(for GHK and G6PI) and the consumption rate of NADH
(for MDH) consecutive to the activity of soluble enzymes
were quantified by spectrometry at 340nm. For measure-
www.biogeosciences.net/10/1155/2013/ Biogeosciences,10,1155–1167,2013
1160 V.Maireetal.: AnunknownoxidativemetabolismcontributestosoilCO emissions
2
70 50 70
A B C
GHK G6PI MDH
60
40 60
50 Total enzyme
A (%)0 2400 SImomluobbleil ieznezdy emnezyme 2300 4500 A (%)0
Ratio A / j 10 YM(ot)d =el :a ·re2 -b=·t 0+. 9c9·e*-*d·*t + f·e-g·t 10 YM(ot)d =e l:a ·re2 -b=·t 0+. 9c9·e*-*d·*t + f·e-g·t YM(ot)d =e l:a ·re2 -b=·t 0+. 9c9·e*-*d·*t + f·e-g·t 2300 Ratio A / j
10
0 0 0
0 10 20 30 40 0 10 20 30 40 0 10 20 30 40
Time (days) Time (days) Time (days)

Fig.2.ActivityofthreespecificenzymesinvolvedinglycolysisandtheKrebscyclefollowingtheirincorporationinthenon-irradiated-soil
fromTheix.E nzymeactivityalongtime(Aj)isexpressedas%oftheinitialactivityofenzymaticsolution(A0)appliedtothesoil.The
activitiesoftoFtaiglu(rdea 2rk circles),soluble(whitecircles)andimmobilisedenzymes(greycircles)aredistinguished.Full,dashedanddotted
linesrepresent thefitofthekineticmodelontotalenzymeactivity,itsconfidenceandpredictiveerrorintervalsat5%P-level,respectively.
(A)G6PI:glucose-6-phosphateisomerase;(B)GHK:glucosehexokinase;(C)MDH:malatedehydrogenase.
2.4.1 Modelofcarbonflux ganisms,duetotheirsize,occupylessthan0.5%ofthesoil
porespace(PaulandClark,1989).
In the model, CO2 emission from non-irradiated soil (Rni) Parameterk variesbetween0–1anddependsonthefrac-
is represented by the sum of living respiration (Rl) and tionofrespiratoryenzymesreleasedbydeadorganismsthat
EXOMET (Rx) (Eq. 2). After irradiation, a fraction k of Rl is stabilised in irradiated-soils. In order to estimate k, we
is converted to EXOMET via the soil stabilisation of respi- incubated the glucose-6-phosphate isomerase (G6PI) in the
ratoryenzymesreleasedbythekilledorganisms.Asaresult, irradiated-soilsofTheix,PontaGrossa,Laqueuille,Sorøand
thesumofEXOMET(Rx)andk·RldeterminesCO2emission Bugac for 14 days. This enzyme was selected because it
fromirradiatedsoil(Ri)(Eq.3).Themodelreadsasfollows showedthesmalleststabilisedfractionamongthethreeres-
piratoryenzymestestedinthesoilfromTheix(Fig.2).The
Rni=Rl+Rx (2) totalG6PI activitywas measuredthroughout theincubation
as described in the Sect. 2.3.2. For each soil, we fixed the
parameter k equal to the fraction of stabilised G6PI activ-
R =k·R +R (3)
i l x ity assuming that the reaction sustained by the G6PI was
the limiting reaction for the EXOMET. Other enzymatic re-
ByfixingthesameR intheirradiatedandnon-irradiated
x actions could be more limiting for extracellular oxidative
soils, the model assumes that irradiation has no effect on
metabolism. In this case, k would have a lower value and
thepre-existentEXOMETRx.Wediscussheretwoexamples the calculated EXOMET would increase which would sig-
where irradiation could modify R . First, the γ-irradiation
x nify that the model underestimates the contribution of the
bydenaturingpartofsoilenzymescoulddecreaseR .Inthis
x EXOMETtosoilCO2emissions.Moreover,parameterkhasa
case,itiseasytoshowthattheEXOMETcontributiontosoil
limitedbearingonthecalculationofEXOMET.Forexample,
CO emissions is underestimated by the model. Neverthe-
2 for the soil from Theix, we calculated that a 50% variation
less,thisunderestimationofEXOMETislikelytobemoder-
ofkcauseda6%deviationinEXOMET.
ate since the effect of γ-irradiation on soil enzymes is typ-
ically low (see review of McNamara et al., 2003). Second,
by suppressing the microbial uptake of organic substrates, 2.4.2 Soilincubationexperiment
the irradiation could increase the availability of these sub-
strates for EXOMET increasing Rx. In this case, the current Experimentalmicrocosmsconsistedof30g(drymassbasis)
modelwouldoverestimatethe EXOMET contributiontosoil samples of fresh sieved soils placed in 250mL flask. Sets
CO2emissions.However,EXOMETandlivingrespirationare of irradiated and non-irradiated soils were prepared for the
notlikelytobeincompetitionfororganicsubstrates.Indeed, five studied soils. Soils were γ-irradiated as previously de-
EXOMET may have preferential access to organic substrate scribed. Soils were incubated in a dark chamber at 30◦C
sinceEXOMET-carryingenzymesareadsorbedonsoilparti- and with a water potential of −100kPa for 21 days. Suf-
clesincludingorganicmatter.Moreover,mostofthesoilmi- ficient soil microcosms were prepared to permit four de-
crositeswhereEXOMETcanproceedarelikelytodeprivedof structive harvests and four replicates per treatment. Micro-
microorganisms. Indeed, enzymes responsible for EXOMET cosms were sampled after 2, 6, 13 and 21 days of incuba-
may diffuse in most soil pores whereas living soil microor- tiontoquantifyCO emissionsandtoverifymaintenanceof
2
Biogeosciences,10,1155–1167,2013 www.biogeosciences.net/10/1155/2013/
V.Maireetal.: AnunknownoxidativemetabolismcontributestosoilCO emissions 1161
2
irradiated-soilsundersterileconditionsthroughouttheincu- 2.7 Dataanalysis
bation.Thesterilitywasverifiedbyacombinationofmeth-
odsincludingelectronmicroscopyandmoleculartracingof AllstatisticaltestswereperformedwiththeStatgraphicsPlus
functionalRNA-producingmicroorganisms(S1).TheCflux software (Manugistics, Rockville, MD, USA). General lin-
model was constrained with CO emissions from irradiated earmodel(GLM)procedures,usingtheLSDmethodinpost
2
(R) and non-irradiated-soils (R ) corresponding to the in- ANOVAmultiplemeancomparisontests,wereemployedto
i ni
cubation period 13–21 days. The 0–13 day period was ex- testeffectsofsoil,treatment(irradiation,substrateandyeast-
cluded because soil stabilisation of respiratory enzymes re- extractamendment,exposuretohightemperature,autoclav-
quiredseveraldays(Fig.2andS3). ingandtoxiccompounds)andtimefactorsonCO2emission
and O consumption. When model residuals did not follow
2
2.5 SomeEXOMETproperties a normal distribution, the variables were log-transformed.
NonlinearregressionswereusedtoanalysekineticsofCO ,
2
Given that we consider EXOMET to be carried out by O concentrationandenzymaticactivity.
2
soil-protected slow-cycling enzymes rather than microbe-
dependent living respiration with tight physiological con-
straints, we predicted that EXOMET would show specific
3 Results
properties. First, we suggest that EXOMET persist in the
long-term(>100days)withoutmicrobialproductionofnew
3.1 DemonstrationofEXOMETbyincubatingacell-free
enzymes. To test this hypothesis, the irradiated soil from
yeast-extractinsterilisedwaterandsoil
Theix was incubated at 30◦C and with a water potential of
−100kPafor332days.TheCO emissionratewasregularly
2 The sterility of microcosms was maintained throughout the
measured during the incubation period in order to calculate
experiment (S1). The emission of CO was null in wa-
2
theEXOMEThalf-life.Second,wesuggestthatEXOMETre- ter with glucose (W+G, data not shown). Despite sterili-
sisthightemperature,pressureandtoxiccompounds.Totest sation, soils with and without glucose (S and S+G) still
this hypothesis, we exposed the irradiated-soil from Theix
emitted CO throughout the incubation (Fig. 3) confirming
to additional treatments: 150◦C for two hours, autoclaving 2
previous observations (Peterson, 1962; Ramsay and Baw-
(137◦Cand2.4105Pafor45min)orchloroformvapoursfor
den, 1983; Lensi et al., 1991; Trevors, 1996). The supply
24h.Allmicrocosmswerethenincubatedat30◦Candawa-
of glucose had no effect on soil CO emissions (S versus
2
ter potential of −100kPa for 21 days. Four replicates were S+G). In contrast, the combined supply of glucose and
prepared for each treatment. The CO emission from soils
2 cell-free yeast-extract containing respiratory enzymes trig-
exposed to temperature, pressure or toxic compounds were
gered sudden and enormous respiration fluxes in water and
comparedtothatofcontrolsoil(onlyirradiated)inorderto soil(W+G+YEandS+G+YE,respectively).TheCO
2
quantifytreatmentseffectsonEXOMET. emissionfromW+G+YEandS+G+YEtreatmentsre-
spectively represented 725 and 72 times that of control soil
2.6 Fluxmeasurements
(after eighteen hours of incubation, Fig. 3). The released
CO originated from added 13C labelled glucose and unla-
In the incubation experiments where gas fluxes were stud- 2
belledsubstratespresentinyeast-extractandsoil(S4).More-
ied (Sects. 2.2, 2.4 and 2.5), two sets of microcosms were
over, CO emissions mirrored the consumption of O for
preparedinordertoquantifygasexchangeandtodetermine 2 2
the 13C content of released CO . In one set of microcosms, alltreatmentsanddates,withtheexceptionofW+G+YE
2
thereleasedCO wastrappedinNaOH.The13Cabundance treatment at eighteen hours of incubation (respiratory quo-
2
tient=2, Fig. 3a). In this particular case, the availability of
ofCO wasanalysedbyIRMSafterprecipitatingcarbonates
2
O wasreduced,metabolicactivitywasintenseandthepres-
withanexcessofBaCl andfiltration.CO andO gascon- 2
2 2 2
sure in microcosms rose to 1.25atm, all of which indicated
centrationsintheothersetofmicrocosmsweremeasuredby
thepresenceoffermentativemetabolism.
gas spectrometry (Agilent 3000µGC, Agilent Technology,
Our results confirm the idea that the enzymatic cascade
Lyon).ConcentrationofCO inatmosphereofflaskswasal-
2
leadingtotheoxidationoforganicC(i.e.,glycolysisandthe
waysmaintainedbelow3%(17%ofO inflaskswhereCO
2 2
Krebscycle)canoccurinanextracellularcontextinsoiland
wastrapped),exceptattwodatesduringtheyeastextractin-
water. This indicates that respiratory enzymes can maintain
cubation experiment. In these latter cases, concentration of
theiractivityoutsidethecellandhaveaccesstosubstrateand
CO reached4%(day10)and22%(day0.75)signifyingthe
2
co-factorfluxtofunction.Itisworthnotingthattheelectron
diffusion of O may have limited the oxidative metabolism
2
transfertoO wasmaintainedsincetheemissionofCO was
(EXOMET)inducedbyyeastextract. 2 2
coupled to the consumption of O (Fig. 3a). This electron
2
transfer, probably carried out by the cytochromes of cellu-
lar debris (Trevors et al., 1982), permits the regeneration of
electronacceptors(i.e.,NAD+)explainingthepersistenceof
www.biogeosciences.net/10/1155/2013/ Biogeosciences,10,1155–1167,2013
1162 V.Maireetal.: AnunknownoxidativemetabolismcontributestosoilCO emissions
2
24 a A Table 2. Size and half-time of fast, intermediate and slow pools
n 20 of respiratory enzymes in the non-irradiated-soil from Theix.
sio 146 a GHK: glucose hexokinase, G6PI: glucose-6-phosphate isomerase,
emis (%) 3 b b a MDH: malate dehydrogenase. Pool size and half-time were ob-
O 2 2 b b tained by fitting the kinetic model presented in Sect. 2.3 to total
C 1 c c c c c b b a c enzymeactivity(Fig.2).
0000
on -1 c c c c c b ba c Fastpool Intermediatepool Slowpool
pti -2 b b Enzyme
onsum (%) ----8434 b b a WS+G+YE a S(%ize) Hal(fm-liinfe) S(%ize) Half-l(ihfe) S(i%ze) Ha(ldfa-lyifse)
O c2 -8 a SS++GG+YE GGH6PKI 3656..84 <<1144 5278..68 286..94 64..68 4331..35
-10
MDH 38.3 <14 47.6 17.1 14.1 495.1
0-0.75 0.75-10 10-19 19-53
Period (days)
100
BB
the soil particles permit EXOMET persistence, possibly by
10 preserving the respiratory enzymes from denaturation and
W+G+YE
n rate cale) 1 SS+G proteolysis.
ssio og s S+G+YE 3.2 Soilstabilisationofoxidativemetabolismenzymes
emi -1d, l 0.1 Kinetic analysis of total enzyme activities indicated the ex-
O 2% istenceoffast-,intermediate-andslow-cyclingpoolsofen-
C (
0.01 zymes in the soil from Theix (Fig. 2, Table 2). Between 36
and 66% of the initial enzymatic activity was lost within
minutesfollowingenzymeadditiontothesoil(thedifference
0.001
0 10 20 30 40 50 60 between initial activity and that 20min after enzyme addi-
Time (days) tion,Fig.2).Thehalf-lifeofthisfastpoolwastoofasttobe

determinedprecisely,butis<14min(Table2).Giventhera-
FFiigg.ur3e. 3( A) Total (labelled+unlabelled) CO2 emission and O2 pidityoftheirinactivation,enzymesofthefast-cyclingpool
consumption from irradiated-soil (S); irradiated-soil+labelled
werelikelydenaturedbyphysico-chemicalprocesses(Burns,
glucose (S+G); irradiated-soil+labelled glucose+yeast-
1982). A second pool of enzymes representing 29–58% of
extract (S+G+YE); water+labelled glucose+yeast-extract
the initial activity was inactivated more slowly with a half-
(W+G+YE) for four periods of incubation. Results are ex-
lifeof9–26h.Soilproteolyticactivitymayhavecontributed
pressed as % of microcosm atmosphere. Water with labelled
tothedegradationofthispool(Sarkaretal.,1989).Finally,
glucose was also incubated as control, but CO2 emission from
this control was null and not reported. Letters at each sampling 5–14% of the initial enzymatic activity was retained in a
dateindicatetheANOVA-baseddifferencesat5%P-levelamong highlystableformwithahalf-lifeof32–495days.Figure2
treatments. (B) Total (labelled+unlabelled) CO2 emission rate showsthatthislong-termpersistenceofenzymaticactivities
fromwaterandsoilmicrocosms.SymbolsarethesameasFig.3a. exclusivelyreliedonenzymesimmobilisedonsoilparticles
(humus and minerals). These results confirm the protective
roleofsoilparticlesforrespiratoryenzymesagainstphysico-
chemicaldenaturationandproteolysis.Ourinvestigationsin
EXOMET over53days(Fig.3).Moreover,ourresultsshow the other soils gave consistent results with 0.8–3.3% of the
thatfermentativemetabolismisanotherintracellularprocess
initialenzymaticactivitystabilised(S2).
thatcanbereconstitutedoutsidethecellwhentheavailability
ofO2fortheEXOMETislow. 3.3 ContributionofEXOMETtosoilrespiration
TheEXOMETwassignificantlyhigherinwaterthaninsoil
duringthefirsteighteenhoursofincubation(W+G+YEver- Despite sterilisation by γ-irradiation, the five studied soils
susS+G+YE,Fig.3b),whichcanbeexplainedbyenzyme released large quantities of CO throughout the incubation
2
inhibitors present in soil (Burns, 1982). However, EXOMET period(Fig.4).After21daysofincubation,cumulatedCO2
continuously decreased with time in water whereas it sta- emissionsfromtheirradiated-soilrepresented17to59%of
bilisedinsoilataraterepresenting247%ofCO emissions that measured in non-irradiated-soil depending on soil type
2
from sterile control soil at day 20. The higher EXOMET in (Fig. 4). The value of model parameter k was determined
soilthaninwaterafter20days(Fig.3b)cannotbeexplained forthesoilsofTheix,PontaGrossaandLaqueuille,butnot
by an exhaustion of C-substrate in water since the second for the soils of Sorø and Bugac where large releases of hu-
glucose dose had no effect (S5). These results indicate that mic acids did not permit the measurement of G6PI activity
Biogeosciences,10,1155–1167,2013 www.biogeosciences.net/10/1155/2013/
V.Maireetal.: AnunknownoxidativemetabolismcontributestosoilCO emissions 1163
2
Table 3. Activity of glucose-6-phosphate isomerase (G6PI) and model quantification of CO2 emissions from living organisms (Rl) and
extracellularoxidativemetabolism(Rx)forthefivestudiedsoils.Rni andRi representCO2 emissionsfromnon-irradiatedandirradiated
soil,respectively,fortheincubationperiod13–21days.k isthefractionofRl convertedinextracellularoxidativemetabolism(EXOMET)
afterirradiation.Contribution(%)ofEXOMETtosoilCO2emissionwascalculatedasRx/Rni·100.nd=notdetermined
Sites
G6PIactivity(%initialactivity) Bugac Laqueuille PontaGrossa Sorø Theix
7days nd 0.5±0.05 5.1±0.4 nd 10.5±0.9
14days nd 0.3±0.03 3.1±0.1 nd 10.0±0.4
ModelquantificationofthetwometabolicpathwaysoforganicCoxidation
Modelinputs
Rni(mgC-CO2kg−1) 168.8±2.6 186.4±3.3 162.8±0.9 200.2±1.5 56.4±0.5
Ri(mgC-CO2kg−1) 98.2±3.2 40.3±1.5 30.6±0.8 34.6±0.9 30.2±0.9
k(%) nd 0.3±0.03 3.1±0.1 nd 10.0±0.4
Modelresults
Rl(mgC-CO2kg−1) nd 146.5 136.4 nd 29.1
Rx(mgC-CO2kg−1) nd 39.9 26.4 nd 27.3
Contributionoftheextracellularoxidative nd 21.3 16.2 nd 48.4
metabolismtoCO2emission(%)
1.4 3.4 SomeEXOMETproperties
oil)
s 1.2 A B C D E Significant CO emissions were maintained throughout the
emissions n irradiated 01..80 a DDaayy 26 ab a 3crea3sl2cuuldtlaacytoeindnfictruhmbastat2ttihhoeenihodafelaifr-rtlhaifdaetiaoEtfeXdEO-XsMoOEilMTfErcoTamnwpTaehsres1iix6st5(iFndiagthy.es5.)lo.TnWhgies-
O 2No Day 13 cc a b termwithoutmicrobialproductionofnewenzymes.Thisper-
ative C d soil / 00..46 b Day 21 cc sleisatseendcebyreaflneccietsntsogielnsetraabtiiloisnastioofnmoifcrroebspiairlaptoorpyuleantizoynms.esre-
el e b Although the treatments applied to the irradiated-soil
R adiat 0.2 cc abcd cc fromTheix(hightemperature,pressureandchloroform)are
rr known to denature unprotected enzymes and be lethal for
(I
0.0 mostmicroorganisms(Koffleretal.,1957;KashefiandLov-
Ponta Grossa Soro Bugac Laqueuille Theix ley, 2003; Rainey et al., 2005; Lopez-Garcia, 2007), signif-
(Brazil) (Danemark)(Hungary) (France) (France)
icant CO emissions persisted during the 21 day incubation
2
F ig. 4. Relative CO2 emission between irradiated and non- period. Based on soil CO2 emissions (Fig. 6), we estimated
iFrirgaudrei a4t edsoilforthefivestudiedsoils.Day2,Day6,Day13and that 50, 20 and 10% of EXOMET were resistant to chloro-
Day 21 represent days of incubation. Letters indicate the differ- form, 150◦C and autoclaving, respectively. Thus, soil par-
ences at 5% level between measurements over time (lower case) ticles not only protect enzymes against denaturation (e.g.,
andacrosssoils(uppercase)basedonrepeated-measuresanalysis
La¨hdesma¨kiandPnspanen,1992),butalsoallowthemainte-
ofvariance.
nanceofcomplexoxidativemetabolisminconditionswhere
lifeisgenerallyimpossible.
(Table 3 and S3). For the three soils where k could be de-
termined,modellingresultsindicatedthatEXOMETsubstan-
4 Discussionandperspectives
tiallycontributedtoCO emissionsfromthenon-irradiated-
2
soils(Table3).EXOMETwasresponsiblefor16,21and48% Our findings show that complex biochemical reactions un-
ofCO emissionsfromthesoilsofPontaGrossa,Laqueuille
2 derpinningrespirationcanoccurinthesoilwithoutcompart-
and Theix, respectively. These unexpected contributions of
mented living structure. Our results also shed light on the
EXOMETindicatethepresenceofalargequantityofrespira- keyrolesofsoilparticles(minerals,soilorganicmatter)im-
toryenzymesinsoilsoutsidecells.
pliedinthisphenomenon.Enzymesinvolvedincelloxidative
metabolismcanmaintaintheiractivityoutsidethecellthanks
www.biogeosciences.net/10/1155/2013/ Biogeosciences,10,1155–1167,2013
1164 V.Maireetal.: AnunknownoxidativemetabolismcontributestosoilCO emissions
2
4,0 well-knownrespirationofsoilbiota,(2)anEXOMETcarried
outbyenzymesreleasedfromdeadorganismsandstabilised
3,5
by soil particles. These key results deserve further experi-
3,0 mentsthatwillverifyassumptionsofourmodeldesignedto
ate -1d) quantify the EXOMET or ideally will propose an alternative
n r oil 2,5 independentmethodforthisquantification.
o s
O emissi2-1mgC kg 12,,50 aCracLtyeicvlylienwgbherecenaspusistreuadttihyoeinnygadneodffnEeocXttslOiokMfeEleyTnvosibhreooyunlmtdoebtnheteaclsoafnamscietdoelrarsewodsnsaetnhpde-
C ( 1,0 respond differently to environmental factors. Soil microor-
Y (t) = 3.68 (±0.09).e-[0.0042 (±0.0003).t ] ganismshavetightphysiologicalconstraintscomprisingspe-
0,5 P < 0.001 r2 = 0.97
cific environmental conditions (temperature, moisture, ab-
half-life = 165 days
0,0 sence of toxic compounds) and needs in energy and nutri-
0 100 200 300
Time (days) ents. These needs explain why soil CO2 emissions are con-

trolledbytheavailabilityoffreshenergy-richCandnutrients

Fig.5.CO2emissionratefromtheirradiated-soilfromTheixincu- tosoilmicroorganisms(Fontaineetal.,2003;Blagodatskaya
bFaigtuerde d5u ring238days.Half-life(t1/2)wascalculatedfromasimple etal.,2007;Pascaultetal.,2013).Incontrast,theEXOMET-
exponentialmodel. carryingenzymeshavefewphysiologicalconstraintsandare
highlyresistanttotoxics,hightemperatureandpressuredue
to their protection by soil particles (section Sect. 3.4). This
150
EXOMET can explain why part of soil CO2 emissions is in-
Irradiated-soil
dependent of microbial biomass size, community structure
125 + CHCl
n 3
sio + 150°C or specific activity (Kemmitt et al., 2008) and is resistant
CO emis2-1kg soil)10705 + Autoclaving thRoiagtmhoxstaeicymcaponemdraBptuoaruwen,ddpesnrea,sn1sd9u8ree3x,;traLenemdnesiriereantdvaiilar.ot,in1om9n9)e1(nP;tTsetr(eecrvhsoloornsr,,o11fo99r96m62;;,
ulated (mgC 50 KtheemflmusithteotfalC.,O2200e8m).isMsioorneofvroemr,thsoeiElsXsOuMbmETittceodutlodefxrepelazien-
m thaw or wet-dry cycles since these treatments promote mi-
u
C
25 crobial death and release of respiratory enzymes in soils
(Henry,2007;BorkenandMatzner,2009;Kimetal.,2012).
0 Finally, the long-term persistence of EXOMET-carrying en-
0 5 10 15 20 25 zymes (Figs. 2 and 5) signifies that current CO emissions
Time (days) 2
from soils partly depend on past microbial activities. This
F ig. 6. Cumulated CO2 emission from the soil from Theix af- memory of soils suggests a delay between the modification
tFeigruerxe p6o suretoirradiation(Irradiated-soil),irradiationand150◦C of microbial activities and its consequence on soil respira-
(+150◦C),irradiationandchloroformfumigation(+CHCl3),and tion.Suchadelaymustbetakenintoaccountwhenstudying
irradiationandautoclaving(+Autoclaving). effects of environmental factors on soil respiration to avoid
anunderestimationofmodificationofsoilfunctioning.
Thecontributionof EXOMET toCO2 emissionswascon-
to the protective role of soil particles. The concentration of trasted among the three soils (Table 3), suggesting an im-
endoenzymesaroundsoilparticlesmayfacilitateexchanges portant role of soil properties and land use on the balance
ofco-substratesandco-factorsbetweenenzymes,whichare betweenlivingrespirationandEXOMET.However,thenum-
necessary for the cascade of reactions implied in oxidative ber of tested soils is too low to draw definitive conclusions
metabolism.Attheendoftherespiratorychain,thetransfer and we can only speculate on some factors that could con-
ofelectronstoO2 maybecarriedoutbysoilparticlesdueto trol the EXOMET. It is interesting to note that the highest
theirelectriccharge(Trevorsetal.,1982). EXOMET contribution to CO2 emissions was found in the
Our results suggest that the EXOMET can substantially soil presenting the highest clay content and the highest ca-
(16–48%)contributetosoilCO emissions.Thisunexpected pacity to stabilise the endoenzyme G6PI activity (soil from
2
contribution of EXOMET suggests the presence of a large Theix, Tables 1 and 3). The high specific area and electric
quantity of respiratory enzymes in soils outside cells. This chargeofclayparticlesmaypromotestabilisationofendoen-
enzymaticpoolmayresultfromthelong-termaccumulation zymesonclaysurface.Theorganic-richsoilfromLaqueuille
of enzymes released from dead organisms and stabilised on has an intermediate position in the ranking of soils accord-
soilparticles(Fig.2).Thus,CO2emissionsfromsoilsareap- ing to EXOMET contribution. This soil curiously presents
parentlydrivenbytwomajoroxidativemetabolisms:(1)the thelowestabilitytostabilisetheendoenzymeG6PIactivity
Biogeosciences,10,1155–1167,2013 www.biogeosciences.net/10/1155/2013/