Rapid abiotic transformation of nitrate in an acid forest soil

16 Pages·2001·0.12 MB·English

Biogeochemistry 54: 131–146,2001.
©2001KluwerAcademicPublishers. PrintedintheNetherlands.
Rapid abiotic transformation of nitrate in an acid
forest soil
DAVIDBRYANDAIL1∗,ERICA.DAVIDSON2&JONCHOROVER1
1DepartmentofAgronomy,ThePennsylvaniaStateUniversity,UniversityPark,PA16802,
U.S.A.;2TheWoodsHoleResearchCenter,WoodsHole,MA,02543,U.S.A.
∗
( authorforcorrespondence,DepartmentofPlant,SoilandEnvironmentalSciences,5722
DeeringHall,TheUniversityofMaine,Orono,ME04469,U.S.A.,
e-mail:[email protected])
Key words: 15N, nitrate, nitrate immobilization, nitrogen cycling, nitrogen deposition,
temperateforests
Abstract. Nitrate immobilization into organic matter is thought to require catalysis by the
enzymesofsoilmicroorganisms.However,recentstudiessuggestthatnitrateaddedtosoilis
immobilizedrapidlyandthisprocessmayincludeabioticpathways.Weamendedlivingand
sterilizedsoilwith15N-labelednitrateandnitritetoinvestigatebioticandabioticimmobiliz-
ation.Wereport rapidtransformation ofnitrateinincubations of theOlayer of forest soils
that have been sterilized to prevent microbial activity and to denature microbial enzymes.
Approximately30,40,and60%ofthe15N-labelednitrateaddedtolive,irradiated,orauto-
claved organic horizon soil disappeared from the extractable inorganic-N pool in less than
15 minutes. About 5% or less of the nitrate was recovered as insoluble organic N in live
andsterilizedsoil,and theremainder wasdeterminedtobesolubleorganicN.Added 15N-
nitrite, however, was either lost to gaseous N or incorporated into an insoluble organic N
formin both liveand sterileorganicsoils. Hence, the fateand pathway of apparent abiotic
nitrateimmobilizationdiffersfromthebetter-knownmechanismsofnitritereactionswithsoil
organicmatter.Nitrateandnitriteadded toliveA-horizon soil waslargelyrecovered inthe
formadded,suggestingthatrapidconversionofnitratetosolubleorganic-Nmaybelimitedto
C-richorganichorizons.Theprocessesbywhichthistemperateforestsoiltransformsadded
nitratetosolubleorganic-Ncannotbeexplainedbyestablishedmechanisms,butappearstobe
duetoabioticprocessesintheorganichorizon.
Introduction
In northeastern North America and throughout much of Europe, the input
of nitrate to forest ecosystems by atmospheric deposition has increased as
muchas5-to-20foldduetofossilfuelburning(Schlesinger &Hartley1992;
Galloway et al. 1995). Initially, this enhanced N input may stimulate forest
productivity. Itappearsthatenhanced Ninputresulted inamodestterrestrial
132
sinkofatmospheric carbon(Houghton etal.1998,Nadelhoffer etal.1999a),
apparently because most of the N from atmospheric deposition remains in
the soil rather than being taken up by plants (Nadelhoffer et al. 1999a,
b). Ultimately, if N inputs exceed demand, limitations on the availability
of other nutrients and cation leaching may result leading to forest decline.
When nitrate inputs exceed the soil nitrate immobilization potential, a state
of N-saturation is said to exist (Aber et al. 1989, 1998; Ågren & Bosatta
1998). Because nitrate immobilization is believed to be mediated biologic-
ally,N-saturation hasbeenrelatedtonitrateinputs,successional statusofthe
vegetation,season,temperature,andavailabilityofothernutrients(Aberetal.
1998).OurabilitytomodelandtopredicttheconsequencesofatmosphericN
deposition depends upon our understanding of the mechanisms of N cycling
withinforestecosystems, including transformations ofnitrate.
In most aggrading temperate and boreal forests, little nitrate is present
in soils because N is conservatively and efficiently cycled within these
ecosystems (Vitousek & Reiners 1975), and because gross rates of nitrate
production via nitrification are generally matched by equal rates of plant
uptake and microbial immobilization (Davidson et al. 1992; Stark & Hart
1997).Exogenoussourcesofnitrateappeartoberetainedprimarilyinorganic
forms. A 15N labeling study at the Harvard Forest in central Massachusetts
showed that only 4–32% of the applied nitrate label was recovered in tree
biomass whereas 51–79% of the nitrate label was found in soil pools at the
end of a single growing season (Nadelhoffer et al. 1999b). A similar result
wasfoundinananalysisofseveralother15N-labelingstudiesinNewEngland
andEurope(Tietema1998; Nadelhoffer etal.1999a). Furthermore, 10years
of fertilizer addition to plots within the Harvard forest at the rate of 150 kg
ha−1 y−1 havesuggestedthatthismid-successional mixedhardwoodforestis
fairlyresilienttolargeincreasesinNavailability,immobilizing>70%ofthe
added nitrogen with at least half of the N retained in the forest floor (Magill
et al. 1997; Magill et al. 2000). However, CO respired as a consequence
2
of organic carbon mineralization, often presumed to be necessary to fuel
microbialNimmobilization(Schimel1988),hasnotbeendetectedfollowing
Nadditions (Aberetal.1998).
Soilmicroorganisms have been shownto immobilize nitrate in temperate
forest soils (Schimel & Firestone 1989; Davidson et al. 1992; Stark & Hart
1997; Tietema & Wessel 1992; Zak et al. 1990), and were the presumed
agents ofnitrate uptake observed inNew England forests (Nadelhoffer et al.
1995).However,recentstudiesof15N-nitrateaddedtoincubationsofHarvard
Forest soils have revealed surprisingly rapid immobilization of the label –
i.e.,disappearance of34–62%of15N-nitratewithin15minutesofaddingthe
label (Berntson & Aber 2000). The degree to which microbially – mediated
133
processes account for such a large and rapid decline in extractable nitrate
is unclear because biotic and abiotic processes could not be assessed inde-
pendentlyintheselivesoilincubations.Rapidnitrateimmobilizationcoupled
with the observation that fertilizer-N is immobilized without concomitant
production ofCO hasfueledspeculation aboutabioticandalternative biotic
2
pathwaysleadingtonitrateimmobilization (Aberetal.1998).
Abiotic reactions of nitrite with soil organic matter (SOM) have been
demonstrated to form organically-bound N (Smith & Chalk 1980; Azhar et
al.1986;Thorn&Mikita2000).Itispossiblethatpartialreduction ofnitrate
to nitrite by biological respiration or by reduced metals could be followed
by abiological reactions of nitrite with SOM. Although there are no known
pathways by which nitrate reacts directly with soil organic matter in abiotic
reactions that result in immobilization, unknown chemical reactions in the
complexmatrixofsoilcannotberuledout.
We incubated O (O ) and A-horizon soils from a hardwood stand at
e/a
the Harvard Forest, Massachusetts, with 15N as either nitrate (NO−) or
3
−
nitrite (NO ) to measure N-immobilization in both sterilized and live soils.
2
The objectives of the study were (i) to investigate the potential for nitrate
and nitrite immobilization; (ii) to distinguish between biotic and abiotic
immobilization; and(iii)toidentifyfatesofimmobilized N.
Materialsandmethods
Soilcollection, characterization andsterilization
The experiment was conducted on O and A-horizon soils collected from the
Harvard Forest, Petersham, MA. Soils were collected from a mixed hard-
wood stand that has been exposed to ambient levels of wet N deposition
estimated to be 0.9 g N m−2 yr−1 with more than 50% as nitrate (Ollinger
et al. 1993; Currie et al. 1996). Soils were sieved (< 2.0 mm), with roots
removedbyhandandthenstoredfieldmoistat4◦Cpriortoanalysis.Organic
C content was 250 g kg−1 for the O and 48 g kg−1 for the A-horizon.
We estimated soil organic-N by submitting whole soil to combustion CHN
analysis and subtracting inorganic-N from this value. Inorganic-N (nitrate,
nitriteandammonium)wasdeterminedcolorimetrically usinga10:1ratioof
0.5mK SO todrysoilextraction at4◦Cfor40min.
2 4
Sterilizationofsoilwasachievedeitherbyautoclaving(2times,3dand1d
priorto15Namendment;121◦Cfor0.5h)orbyγ-irradiation(finaldosage4
Mrad)ofseparatesoilsamples.Acomparisonofthetwosterilizationmethods
wasperformed because neither isfreeofartifactual effectsonsoilproperties
(Ramsey & Bawden 1983; Lotrario et al. 1995). Irradiation at doses higher
134
thanthoseusedinthisstudyhavebeenshowntoproducenitrite,andthisster-
ilization method does not completely inhibit extracellular enzymatic activity
(Cawse& Crawford 1967). Autoclaving increases soluble C and Nfractions
and also denatures intracellular and extracellular enzymes. The effects that
each technique hadon selected soil properties measured inthe present study
arereportedinTable1.Maintenanceofsterilityoverthecourseofincubation
wasconfirmedbyelimination ofCO production andnegativestandardplate
2
counts(i.e.,nocolonyformingunits)forsoildilutions(10−1 to10−6),plated
onmultiplenutrient media.SolublesoilC(DOC)wasmeasuredonacidified
10:1 water to soil extracts using a Shimadzu Model 5000A Total Organic C
Analyzer.
Inorganic15Namendments
Initial soil nitrate and nitrite contents were below our limits of detection
(using 10:1 extracts; limits of 0.4 and 0.2 µg N per gram soil, respectively).
We added filter sterilized 15N (99% atom enriched) nitrate or nitrite at the
rate of 5 µg N per gram soil dry mass, dissolved in a solution that brought
the soil to 50% of water holding capacity as determined on non-sterilized
soils. Soil and label were mixed aseptically with a sterile glass rod, which
wasthenenclosedwiththesoilina350mLincubationjar.Eachcombination
ofsoiltreatment(‘live’control,irradiatedsterile,andautoclavedsterile),and
Namendment (15N-nitrate, 15N-nitrite) wasreplicated three times. Jars were
incubatedfor24hat20◦ C.Sub-samplesweretakenimmediately(<5min)
and24hafter15Naddition forNpoolfractionation.
At each sampling time a 4 g sub-sample of soil from each replicate was
removed to a 50 mL centrifuge tube, closed and placed upright in a bath of
90% ethanol and dry ice. This freezing bath ceased microbiological activity
andpreparedsamplesforfreezedryinginpreparation formassspectrometry.
A second 4.0 g soil sub-sample was added to a 50 mL centrifuge tube
containing20mLcold(4◦C)0.5mK SO andshakenvigorouslyfor20min
2 4
attherefrigeratedtemperature.Thesuspensionwascentrifuged(>25,000g)
to sediment soil and microorganisms into a pellet. The supernatant solution
was removed by aspiration and then filtered (0.45 µm pore size). A second
extraction of the soil pellet was performed as above. The soil light fraction
(thatmaterial notpelleted undercentrifugation) wasreturned tothepelletby
removing it from the surface of the filter. This pellet was then freeze-dried
andprepared formassspectrometry, theresultant15Nreferredtohereafteras
insoluble-N, astheK SO extractionprocessdidnotremoveitfromthesoil.
2 4
Foreach replicate thetwofiltered extracts werecombined (toachieve afinal
− − +
extractant to soil ratio of 10:1) and NO , NO , and NH were determined
3 2 4
colorimetrically <1dafterextraction.
135
A 1.0 g soil sub-sample was dispersed in 0.2% sodium pyrophosphate
solution(pH6.0)andseriallydilutedtoperformviableorganismplatecounts.
Soilsamplesremovedatthebeginning andendofthe24hincubation period
were serially diluted to 10−1, 10−3 and 10−6 dilutions and these dilutions
were used to inoculate reduced-strength trypticase soy agar (TSA) and soil
isolation agarculture plates (Martin1975;Trolldenier 1996). Comparisonof
platecountsamongthepretreatments isreported inTable1.
Givenamaximumtimeforremoval,weighing, extraction, peroxide treat-
ment, or freezing, no ‘initial’ soil sample was incubated with label for more
than15minutes.
15Npoolenrichmentdetermination
Total 15N recovery after incubation was determined by submitting freeze-
dried soil samples directly to isotope ratio mass spectrometry analysis. An
ammonia diffusion method was used to trap inorganic-N on acidified filter
disks to prepare them for isotope ratio mass spectrometry (Stark & Hart
+
1996). K SO extracts were diffused sequentially to obtain NH and then
2 4 4
− −
NO /NO onacidifieddisksusingamodified(Sigmanetal.1997)procedure
2 3
ofSørensen and Jensen (1991). 15Nstandards and blanks wereprepared and
isotope diffusion bias correction calculations were performed as outlined in
StarkandHart(1996) forboth inorganic-N samples. SoilNpoolenrichment
isreported asapercentofinitiallabelrecoveredamongthevariousNforms:
total15Nrecovered, nitrateplusnitrite, ammoniumandorganic-N.
Duringthecourseofanalysisitbecameevidentthatforsamplesincubated
with 15N as nitrate, the 15N in inorganic pools (NO−/NO− and NH+; pools
2 3 4
c and d in Figure 1), and insoluble N in the pellet (pool b, Figure 1) did
not sum to that 15N recovered in the non-extracted soil (pool a, Figure 1),
e.g.,a>b+c+d.Thiswasnotthecase for15N-nitrite incubated soils, where
a=b+c+d.Massbalance calculations indicated that30to60%ofNwasnot
accountedforinsamplesincubatedwith15NO−.Theonlynon-measured soil
3
pool of N was the organic N removed by the K SO extraction, which we
2 4
refer to as dissolved organic N (DON). We then utilized archived samples
to measure the DO15N pool directly, which greatly improved the mass
balance and confirmed the fate of 15NO−. Samples were extracted with 0.5
3
− −
m K SO as before and the K SO extracts were analyzed for NO /NO ,
2 4 2 4 2 3
NH+,concentrations and15Nenrichmentasdescribedabove(Figure1).Pool
4
sizes and 15N enrichment of these new extracts were compared to those of
previousanalysesoffreshsoilandextractsinordertoaccountforanychanges
broughtaboutbyfreeze-dryingandstorageofsamples.Nodifferencesdueto
freeze-drying orstorageweredetected. DissolvedorganicnitrogeninK SO
2 4
soil extracts was measured by the persulfate oxidation method (D’Elia et al.
136
Figure1. Procedurefordeterminationof15NenrichmentofsoilNpools.
+
1976; Ameel et al. 1993). After oxidation of the DON and dissolved NH
4
−
toNO ,wediffused this solution toacidified disks todetermine directly the
3
enrichmentoftheDONpoolbymassspectrometry (MS)(poole,Figure1).
Results
Effectsofsterilization treatmentonsoils
Autoclaving, and to a lesser degree γ-irradiation, increased the soluble or
K SO -extractable amounts of organic-C and ammonium in both O and A
2 4
soils (Table 1). Soluble organic carbon in the O soil, reported as DOC,
increased from < 0.2 g C kg−1 soil in control soil to 1.5 g C kg−1in irra-
137
Table1. Soilpropertiesandeffectsofsterilizationtechniquesonsoilchemistry
Treatment pH DOCα NO−β NH+ OrganicN Platecountsδ
3 4
(g/kg) (mg/kg) (mg/kg) (mg/kg) CFU/g
O-horizon
Control 3.9 0.16(0.02) nd 16(0.37) 10,800(415) 3.2×105
Irradiated 4.0 1.58(0.02) nd 95(5.14) 11,400(274) ng
Autoclaved 3.8 5.38(0.45) nd 119(6.67) 12,000(1013) ng
A-horizon
Control 4.3 0.06(0.01) nd+ 1.41(0.64) 2,400(88) 1.1×105
Irradiated 5.0 0.89(0.01) nd 18.07(0.14) 2,400(50) ng
Autoclaved 5.0 0.96(0.04) nd 43.17(0.94) 2,460(149) ng
αWaterextractableorganiccarbon.
βNO− = (NO− + NO−); nd = not detected, limits of detection 0.04 mg N/L in a 10:1
3 3 2
solution-to-soil0.5MK SO extraction.
2 4
δ10−1 and 10−6 serial dilutions of 10:1 water-to-soil slurries plated on TSA and Soil
◦
Isolation Agar and incubated for 5 d at 20 C. CFU = Colony Forming Units at day 5
onapergsoildrymassbasisaveragecalculatedfromplateswith30–300 cfu’s;ng=no
growthobserved.
diated and > 5 g C kg−1 in autoclaved soils. Nitrate and nitrite remained
undetectable (prior to 15N addition). An increase in soluble ammonium was
observed, presumably from physico-chemical mineralization of the organic-
Npool.ThepHofA-horizonsoilwasslightlyincreasedinirradiatedsoiland
autoclaved soil;nodifferences weredetectedinOsoil(Table1).
Fateofinorganic15Naddedtosoil
A-horizon soil. Nearly 100% of 15N added as either NO− or NO− to
2 3
live control A-horizon soil was recovered in the form added immediately
after addition (Table 2). In contrast, immediate losses of added 15NO−
2
were observed in the autoclaved and irradiated A-horizon soil. Losses of
15NO−increased during the 24-hour incubation in all three pretreatments
2
(Table2),andthiswasattributedtogaseouslossasthe15Nwasnotrecovered
in whole soil submitted to isotope ratio MS. Losses could have been due to
−
microbial or abiotic reduction of NO to N gases (Firestone & Davidson
2
1989),whichwerenotmeasuredinthisstudy.
The recovery of 15NO− also declined somewhat over 24 hours in both
3
controlandautoclavedA-horizonsoil,butthedifferencebetweencontroland
sterile soils could not be evaluated statistically due to a loss of two of three
replicates (Table2).About12%ofadded nitratewasrecovered asorganic-N
incontrolsoils.Recoveryof15N,addedaseitherNO− orNO−,inorganic-N
2 3
138
Table2. Fateof15N-nitrate,nitriteinA-horizonsoil:%15Nrecoveredintheformadded
immediatelyafteradditionandafter24h
Soilpretreatment 15NO−amended 15NO−amended
2 3
t=15min. t=24h t=15min. tv=24h
Control 92.4±4.0 22.7±1.9 101.6±16.0 82.2±ND
Irradiated 40.5±11.5 7.4±0.6 79.3±7.0 75.8±2.2
Autoclaved 14.6±9.9 3.9±1.7 90.1±16.0 64.4±12.0
%Recoveryof15Nreportedasameanof3samples±SD.
ND=SDnotdetermined;1of3sampleswasdetermined.
+
orNH poolsofA-horizonsoilwaslessthan2%inirradiatedandautoclaved
4
soils(datanotshown).
O-horizon soil: nitrite addition. Total recoveries of 73% to 85% of added
−
NO wereobservedinO-horizoncontrolsoils(Figure2).Almosthalf(40to
2
48%)ofthe15Nwasrecovered intheextracted pellet, suggesting substantial
interaction between 15NO− and soil organic matter producing an insoluble
2
−
organic-N product (Figure 2). Theimmobilization ofNO inO-horizon soil
2
occurred within minutes of addition to both sterile and live samples, and no
significantchangesoccurredoverthecourseofthelivesoilincubation(Figure
2(a)). Upto2.5% of15N-NO− wasrecovered asNH+ inlivesoil and lessin
2 4
sterilized soil(Figure2).
Total recovery of 15N in NO−-amended irradiated and autoclaved O-
2
horizon soil was < 50% and < 25%, respectively (Figure 2(b), (c)). The
reduced recovery (as compared to live soil) was detectable in samples taken
immediatelyafter15NO− addition. Incontrast to15NO− amendedA-horizon
2 2
soils,recovery intheOhorizon didnotdecrease significantly duringthe24h
incubation. Aswas the case for the A-horizon soils, the rapid disappearance
−
ofNO addedtoOhorizonsoilisattributedtogaseousloss.Greaterthanhalf
2
of the recoverable 15NO− in sterilized soil was in the insoluble organic pool
2
oftheextractedpellet,andtheremaining15Nwasrecoveredlargelyasnitrite
(Figures 2(b), 2(c)). The sum of 15N recovered as insoluble organic-N plus
−
thatremainingintheNO poolwasroughlyequaltothatrecoveredwhenthe
2
non-extracted wholesoilwassubmitteddirectly for15Nanalysis (Figure2).
−
O-horizon: nitrate addition. Recovery of 100–120% of NO immediately
3
after addition to organic horizon soil was calculated for all treatments when
non-extracted soils were submitted directly to isotope ratio mass spectro-
metry (Figure 3). Recoveries > 100% could be due to analytical errors or
−
to an actual amendment rate of NO somewhat greater than the target of 5
3
139
Figure 2. Percent recovery of 15N in O-horizon soil N pools reported for (a) control, (b)
gammairradiatedand(c)autoclavedsoilsamendedwith15NO− attherateof5µg15Nper
2
gramsoildrymass(n=3;±SD).TotalN=soil15Nrecoveredindirectmassspecanalysis
of non-extracted soil.Org-N Insolb. =15N remaining insoil pelletafter K SO extraction.
2 4
Columnswithdifferentuppercaselettersindicatesignificantdifferencewithin-pool,between
soiltreatmentsatα=0.05level.Columnswithdifferentlowercaselettersindicatesignificant
differencebetweensoilNpoolswithinasoiltreatmentatα=0.05level.
140
µg N g−1. Less than 1% of 15N was recovered as NH+. About 60% of the
4
added 15NO− was recovered as NO− with no significant change from initial
3 3
to final sampling for all soil pretreatments. About 5–8% of the label was
recoveredasinsoluble organic-N intheextracted pellet.Greaterthan80%of
the remaining 15N was recovered as soluble organic N (DON) in persulfate
digestsofK SO extractsoftheOhorizonsoils.Roughly30,40and55%of
2 4
added15NO−tocontrol,irradiatedandautoclavedsoilswasrecoveredinthis
3
DONfraction.
Discussion
Recentreportsofanappreciable capacity fornitrateimmobilization inforest
−
soils subjected to high NO deposition and excess N by experimental fertil-
3
ization are difficult to explain solely by biological mechanisms (Aber et al.
1998;Magilletal.1997;Berntson&Aber2000).Inordertoexaminemicro-
bial and abiotic N immobilization, we chose two independent methods to
−
sterilizesoils.AlthoughNO istheformofNinatmosphericdeposition that
3
−
we are studying, we also included studies of NO immobilization to see if
2
− −
NO andNO havesimilarfates.Similarfatesmightindicatethatbiological
3 2
− − −
reductionofNO toNO isfollowedbysubsequentabioticreactionsofNO
3 2 2
withsoilorganicmatter(Azharetal.1986).
Fateof15N-nitriteaddedtosoil
−
We observed a high potential for reaction of NO with viable O and A
2
horizonsoils.Upto77%of15NO−addedtoA-horizonsoilwaslost,presum-
2
ablytogaseous-N,within24hofaddition.GaseouslossofNwasexacerbated
−
by sterilization; both irradiation and autoclaving of soils prior to NO addi-
2
tion increased N loss markedly. Rapid production of large pulses of NO and
N Ohavebeenobservedwhennitritewasaddedtosterilizedsoils(Davidson
2
1992), and spontaneous self-decomposition or dismutation of nitrous acid
−
(HNO ) to NO and/or NO has been reported for acid soils with pH values
2 2
similar to those used in this study (Chalk & Smith 1983, Schwartz & White
1983). Of the label that was not lost to gaseous processes, nearly all was
−
recoveredasNO inAhorizon soils(Table2),butupto50%wasrecovered
2
asinsolubleorganic-N immediatelyuponadditiontoO-horizonsoils(Figure
2).Hence,reactionwithorganicmatterappearstocompetewellwithgaseous
lossprocessesinsoilwithhigherorganicmatter.Thesefindingsareconsistent
−
with investigations showing increased NO fixation capacity in soils with
2
high organic matter content and pH less than 5 (Nelson & Bremner 1969,
1970).