Q8

Q3

Q2

Q1

lable at ScienceDirect

Soil Biology & Biochemistry xxx (2014) 1e7

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354

55

SBB6047_proof ■ 1 December 2014 ■ 1/7

Contents lists avai

Soil Biology & Biochemistry

journal homepage: www.elsevier .com/locate/soi lb io

56575859606162636465666768697071

Carbon mineralization is promoted by phosphorus and reduced bynitrogen addition in the organic horizon of northern hardwood forests

Melany Fisk*, Sharon Santangelo, Kevan Minick 1

Department of Biology, Miami University, Oxford, OH, 45056, USA

7273 74757677787980818283848586

a r t i c l e i n f o

Article history:Received 8 August 2014Received in revised form18 November 2014Accepted 21 November 2014Available online xxx

Keywords:Forest floorMicrobial nutrient limitationNorthern hardwood forestC mineralizationP limitation

* Corresponding author. Tel.: þ1 513 529 3181.E-mail address: fiskmc@miamioh.edu (M. Fisk).

1 Present address: Department of Forest Resourcestion, Virginia Tech, Blacksburg, VA, 24060, USA.

http://dx.doi.org/10.1016/j.soilbio.2014.11.0220038-0717/© 2014 Published by Elsevier Ltd.

878889909192

Please cite this article in press as: Fisk, M., eorganic horizon of northern hardwood fore

a b s t r a c t

Limitations to the respiratory activity of heterotrophic soil microorganisms exert important controls ofCO2 efflux from soils. In the northeastern US, ecosystem nutrient status varies across the landscape andchanges with forest succession following disturbance, likely impacting soil microbial processes regu-lating the transformation and emission of carbon (C). We tested whether nitrogen (N) or phosphorus (P)limit the mineralization of soil organic C (SOC) or that of added C sources in the Oe horizon of succes-sional and mature northern hardwood forests in three locations in central New Hampshire, USA. Added Nreduced mineralization of C from SOC and from added leaf litter and cellulose. Added P did not affectmineralization from SOC; however, it did enhance mineralization of litter- and cellulose- C in organichorizons from all forest locations. Added N increased microbial biomass N and K2SO4-extractable DONpools, but added P had no effect. Microbial biomass C increased with litter addition but did not respondto either nutrient. The direction of responses to added nutrients was consistent among sites and betweenforest ages. We conclude that microbial N limitation promotes mineralization of C from SOC, whereas Plimits mineralization of C from new organic inputs. We also suggest that N suppresses respiration inthese organic horizons either by relieving the N limitation of microbial biomass synthesis, or by slowingturnover of C through the microbial pool; concurrent measures of microbial growth and turnover areneeded to resolve this question.

© 2014 Published by Elsevier Ltd.

9394

95 96979899

100101102103104105106107108109110

1. Introduction

The activities of soil heterotrophs process plant-derived organiccarbon (C), determining its pathway back to the atmosphere as CO2or into stabilized forms that could persist in soil pools for manyyears. The controls of microbial respiratory activity thus are of in-terest for understanding patterns of soil C storage, as well asnutrient mineralization, in different ecosystems. Evidence exists fornitrogen (N) limitation (Schimel and Weintraub, 2003; Meiduteet al., 2008; Kamble and Bååth, 2014) and also for phosphorus (P)limitation (Cleveland et al., 2002, Cleveland and Townsend, 2006;Craine et al., 2007; Bradford et al., 2008) of microbial growth andactivity in forest soils. While some evidence supports the idea thatforest productivity and microbial respiration are limited by the

& Environmental Conserva-

111112113114115116

t al., Carbon mineralizationsts, Soil Biology & Biochemist

same nutrient (Reed et al., 2011), this is not always the case and itremains generally unclear what determines patterns of limitationin individual ecosystems. Understanding nutrient limitation ofrespiratory activity is further complicated in some ecosystems byits dependence on the form of C (soil organic C (SOC) or new inputsof C) that is metabolized (Reed et al., 2011).

In this study we examined nutrient controls of microbialrespiration, as part of efforts to understand nutrient limitation innorthern hardwood forests (Vadeboncoeur, 2010; Fisk et al., 2014).These controls are of interest for understanding interactions be-tween nutrient availability processes and soil C storage in responseto ecosystem management or environmental change. In north-eastern hardwood forests, acidic deposition has enriched soils withN and depleted essential cations, such as calcium (Ca) (Lawrenceet al., 1995; Fenn et al., 1998; Likens et al., 1998; Aber et al.,2003), and potentially affected the availability of P via decliningsoil pH (Carreira et al., 2000; Fiorentino et al., 2003). Forest harvestalso alters ecosystem nutrient status via nutrient removal andchanges in recycling processes as vegetation recovers and matures(Fisk et al., 2002; McLauchlan et al., 2007; Milcu et al., 2011;

117118119

is promoted by phosphorus and reduced by nitrogen addition in thery (2014), http://dx.doi.org/10.1016/j.soilbio.2014.11.022

Fig. 1. Locations of the Bartlett Experimental Forest (BEF), Hubbard Brook Experi-mental Forest (HB), and Jeffers Brook site (JB) within the White Mountain NationalForest, NH, USA. Plot layouts are shown within each site; plots sizes are not to scale inthe BEF. One composite sample was analyzed from each plot. Samples from shadedplots were used for the litter and nutrient addition experiments (C8 was not included);samples from shaded plots in C9 and C4 were used for the cellulose and glucoseaddition experiment.

M. Fisk et al. / Soil Biology & Biochemistry xxx (2014) 1e72

1234567891011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556575859606162636465

66676869707172737475767778798081828384858687888990919293949596979899

100101102103104105106107108109110111112113114115116117118119120121122123124125126127128129130

SBB6047_proof ■ 1 December 2014 ■ 2/7

Rastetter et al., 2013). These anthropogenic effects take place on atemplate of substantial landscape-level and regional variation insoils and microbial processes in these northern hardwood ecosys-tems (Johnson et al., 2000; Bohlen et al., 2001; Fisk and Fahey,2001; Fisk et al., 2010).

Our goal in this study was to test whether N or P limit themineralization of organic C in northern hardwood forest soils in thenortheastern US, and whether effects differ in response to added Csources. We incorporated both spatial and successional sources ofvariation in our study to examine the generality of the microbialresponse to nutrient additions, by working in mid-successional andmature forests, and in three separate northern hardwood sites incentral New Hampshire, USA. We focused on the surface organichorizon (Oe), where microbial processes can be crucial limitingsteps in C transformations in these northern forest ecosystemsbecause of the absence of vertical mixing by large decomposerorganisms. Leaching and fungal translocation are the primaryshort-term means of downward vertical movement from surfacehorizons (Frey et al., 2003; Dittman et al., 2007). Most organic C ofplant structural origin therefore decomposes in place, and is eithermineralized to CO2 or is transformed into dissolved forms or thehumus of the Oa horizon. Thus, controls of respiratory processes inthis horizon are crucial for understanding microbial contributionsto patterns of soil C storage.

2. Materials and methods

2.1. Study sites and sample collection

We studied northern hardwood forests in three sites in centralto southeast New Hampshire, USA (Fig. 1). Three mid-age(30e36 yrs since clearcut) stands and three mature (>100 yrsold) stands were in the Bartlett Experimental Forest (BEF;44�2e40N, 71�9e190W; elevation 250e500 m). One mid-age andone mature stand were in the Hubbard Brook Experimental Forest(HBEF; 43�560N, 71�440W; elevation 500 m), and one mid-age andone mature stand were in the Jeffers Brook forest (JB; 44� 020 N,71� 530 W; elevation 730 m). Soils are mostly spodosols (typic andaquic haplorthods) that developed in glacial till derived fromgranitic rock at BEF, from a mix of granitic rock and mica schist atHBEF, and from amphibolite at JB. The mature forest is generallydominated by American beech (Fagus grandifolia Ehrh.), yellowbirch (Betula alleghaniensis Britt.), and sugar maple (Acer saccha-rum Marsh.), with some ash (Fraxinus americana L), red maple(Acer rubrum L), and white birch (Betula papyrifera Marsh). Themid-age stands originated following clearcut harvest and forestcomposition is typical of early-successional hardwood forests ofthe region, with pin cherry (Prunus pensylvanica L.f), white birch,yellow birch, red maple, and American beech common in theoverstory. In each forest stand we sampled soils in three50 � 50 m plots, except for the mid-age plots at JB and HBEF,which were 30 � 30 m. At BEF, these were the same plots asdescribed in Naples and Fisk (2010).

Multiple soil cores (approximately 30; 2 cm diam) werecollected from each plot in early June, 2008. The Oe horizon wasseparated from the underlying Oa by visual criteria in the field. Weidentified Oe as the layer of decayed organic matter that was ofrecognizable plant origin or was fibrous and reddish to dark brown.We separated this from the non-fibrous amorphous organic matterof the Oa that was closer to black in color, had a smooth or greasyconsistency, and sometimes contained mineral flecks. Oe sampleswere pooled into one composite sample per plot. Soils were storedat approximately 4

�C prior to processing (within one week) and

thenwere homogenized gently by hand to remove roots and coarsefragments and to facilitate uniform subsampling.

Please cite this article in press as: Fisk, M., et al., Carbon mineralizationorganic horizon of northern hardwood forests, Soil Biology & Biochemist

2.2. Mineralization and microbial biomass

We quantified C mineralization, N mineralization, and microbialbiomass C (MBC) and N (MBN) in laboratory incubations of allsamples (3 plots per stand � 10 stands). From each sample, an“initial” subsample (10 g freshmass; 4e5 g drymass) was shaken in100 mL 2 M KCl, allowed to settle for approximately 16 h, andfiltered throughWhatman #1 filter paper. A second subsample wasincubated for 21 d at 20e23 �C in a canning jar. After incubation,soil in each jar was further divided into 3 subsamples. One sub-sample of incubated soil (“final”) was extracted in 2 M KCl in thesame manner as the initial subsample. We used a phenolate-hypochlorite method to quantify NH4

þ (method 351.2, US EPA1983) and a cadmium reduction method to quantify NO3

- (method353.2, US EPA 1983) in extracts. Net N mineralization potential wasestimated as the difference in extractable NH4

þ þNO3� between final

and initial extracts.The remaining subsamples of incubated soil were subjected to

the chloroform-fumigation extractionmethod (Brookes et al., 1985)to quantify MBC and MBN. One subsample was shaken in 0.5 MK2SO4 for 30 min and filtered immediately through Whatman #42paper. The other subsample was fumigated with chloroform for 3 din a humid desiccator, and then extracted in the samemanner. For Nanalysis, extracts were subjected to persulfate digestion and NO3

� indigests was quantified using the above cadmium reductionmethod.Carbon in extracts was quantified using a Schimadzu TOC analyzer.MBC andMBNwere estimated as the difference between fumigatedand unfumigated extracts, correctedwith Kc of 0.45 for C and a Kn of0.54 for N (Brookes et al., 1985; Vance et al., 1987).

We estimated C mineralization by quantifying CO2 evolved fromsoils throughout the 21 d incubation. Glass scintillation vials

is promoted by phosphorus and reduced by nitrogen addition in thery (2014), http://dx.doi.org/10.1016/j.soilbio.2014.11.022

M. Fisk et al. / Soil Biology & Biochemistry xxx (2014) 1e7 3

1234567891011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556575859606162636465

66676869707172737475767778798081828384858687888990919293949596979899

100101102103104105106107108109110

SBB6047_proof ■ 1 December 2014 ■ 3/7

containing 10 mL 0.1 M NaOH were sealed in jars and continuouslyreplaced at 40e100 h intervals. The NaOH in these vials was titratedin the presence of a saturating concentration of BaCl2 to quantifythe amount of CO2 that had reacted with NaOH.

2.3. Nitrogen, phosphorus, and carbon treatments

We tested effects of added N and P on the mineralization of Cfrom several sources (SOM, leaf litter, cellulose, and glucose) in twoseparate sets of laboratory incubations, conducted in parallel withthose of untreated soils. In the first set of laboratory incubations weused soils collected from one plot in each stand except for one ofthe mature stands at BEF (9 plots total; Fig. 1). We added leaf litteras the C source and quantified the response by C and N minerali-zation and microbial biomass C and N. Five replicate subsamplesfrom each sample were weighed into canning jars and receivedadded N, P, leaf litter, litter þ N, or litter þ P. Litter treatmentsreceived 0.4e0.5 g of ground sugar maple litter that was collectedfrom nearby the HBEF site. Litter additions equaled 10% of soil drymass and added 0.79 mg N/g soil and 0.034 mg P/g soil.

Nutrient treatments were added in solution in ultrapure water(1 mL/g soil to bring soils to a water content averaging 350%; closeto field capacity). Nitrogen treatments received 2mg N/g soil (equalto 2.5 mg N/g organic matter (OM)) as NH4NO3, and P treatmentsreceived 0.67 mg P/g soil (equal to 0.84 mg P/g OM) as NaH3PO4).Oe horizons average approximately 1280 g OM/m2 in our studysites; if fertilizer were retained in this horizon then these levels of Nand P are equal to fertilizationwith about 3.2 g N/m2 and 1.1 g P/m2,comparable to current levels of fertilization (3 g N m�2 yr�1 and1 g P m�2 yr�1) applied in an NXP experiment in our study sites(Fisk et al., 2014). Untreated soils and litter only treatment receivedthe same quantities of ultrapure water as did nutrient treatments.Solutions and water were added dropwise to the soil surface. Allsoil subsamples were gently mixed after treatment additions andincubated in sealed canning jars for 20 d at 20e23

�C. Carbon and N

mineralization and MBC and MBN were quantified as describedabove.

In the second set of laboratory incubations we quantified effectsof N and P on mineralization of C from added cellulose and glucose,in Oe horizon subsamples from one plot in a mature stand (C9) anda mid-age stand (C4) in BEF (Fig. 1). Seven replicate subsamplesfrom each stand were weighed into canning jars and received thefollowing treatments: control, glucose, cellulose, and each Csource þ N or þ P. Incubations received 8 mg C/g soil, 1 mg N/g soil(as NH4NO3), and 0.33 mg P/g soil (as NaH3PO4). Glucose and

Table 1Carbon (C) and nitrogen (N) mineralization (mg g�1 d�1) and microbial biomass (mg C or NNew Hampshire, USA. Standard errors of the mean are in parentheses.

C mineralization Net Nmineralization

DOC

Bartlett experimental forestMid-age 347 (27.7) 7.3 (0.84) 139Mature 349 (28.6) 2.3 (0.33) 138Hubbard brook experimental forestMid-age 340 (69.0) 6.2 (1.33) 148Mature 316 (67.3) 5.1 (1.12) 156Jeffers brookMid-age 236 (43.5) 4.2 (2.88) 740Mature 260 (24.0) 2.3 (1.72) 920ANOVA resultsSite: F, 4.50, 0.43, 2.34P 0.02 0.68 0.21Age: F, 0.03, 0.21, 0.50P 0.86 0.67 0.52Site � age: F, 0.19, 0.01, 1.74P 0.83 0.99 0.29

Please cite this article in press as: Fisk, M., et al., Carbon mineralizationorganic horizon of northern hardwood forests, Soil Biology & Biochemist

nutrients were added in solution and each soil subsample receivedthe same amount of water, approximately 1 mL/g soil to bring soilsnear field capacity. Cellulose was added as a fine powder mixedwith talcum to facilitate even distribution. Soils were incubated insealed canning jars for 21 d at 20e23

�C and C mineralization was

quantified as described above.Data were analyzed using Proc Mixed in SAS 9.2 (Cary, NC). Ef-

fects of forest age and site on mineralization and microbial biomasswere tested using a mixed-effects model with forest age (mid-age,mature) and site (BEF, HBEF, JB) as fixed effects and stands nested asrandom effects within age. We tested the effects of the interactionof C source and either N or P on C mineralization, MBC, MBN, DOC,and DON in mixed effects models with additional fixed effects oflitter and nutrient (either N or P) treatment. Interactions betweennutrient effects and age or site were not found for C mineralizationor microbial biomass C or N and were therefore not included in thefinal model. Differences among treatments were tested with Tukeydifferences of least square means. Repeated measures were addedto the model for analyzing C mineralization rates over time.

3. Results

3.1. Mineralization and microbial biomass

In control soils, C mineralization and microbial biomass Cdiffered among sites but not between forest ages (Table 1). DOCappeared to follow the same pattern among sites, although differ-ences were not significant, and was correlated with C mineraliza-tion (R ¼ 0.86, P ¼ 0.003). No age or site effects were detected for Nmineralization, DON, or microbial biomass N (Table 1).

3.2. Treatment responses: C mineralization

Rates of C mineralization in control soils varied among standsfrom 250 to 320 mg g�1 d�1 but followed no consistent trend overtime. Added leaf litter increased rates of C mineralization (Table 2),causing a flush of C mineralization over the first 2 d that more thandoubled control rates. Rates in the litter treatment dropped toapproximately half of their maximum over the next 3 d and thendeclined very slightly over time for the remainder of the incubation(Fig. 2A).

Adding N significantly reduced rates of C mineralization(Table 2). In the absence of leaf litter added N had little effect in thefirst 2 d, after which it reducedmineralization of SOC by 31% (HBEF)to 38% (JB) compared to controls (Fig. 2A). Nitrogen added with

/g) in mid-successional and mature northern hardwood forests in different sites inQ6

DON MBC MBN

0 (135) 311 (6.0) 10840 (873) 913 (186.7)0 (64) 211 (41.2) 9170 (1014) 825 (79.2)

0 (72) 425 (17.3) 7760 (1147) 748 (82.0)0 (93) 331 (53.7) 10770 (2021) 830 (237.4)

(139) 248 (81.4) 6530 (1103) 608 (61.1)(171) 291 (82.9) 7700 (498) 803 (52.6)

1.53 8.64, 0.340.24 0.04 0.73

, 0.26 0.37, 0.120.62 0.58 0.74

, 1.06, 0.21 0.280.36 0.82 0.77

111112113114115116117118119120121122123124125126127128129130

is promoted by phosphorus and reduced by nitrogen addition in thery (2014), http://dx.doi.org/10.1016/j.soilbio.2014.11.022

Table 2Anova results (F ratios and P values) for the mixed effects model testing the leaf litter � nutrient addition interaction effects on C mineralization (with repeated measures),extractable DON and DOC and microbial biomass pools.

Source C mineralizationon DON DOC MBN MBC Microbial C:N

F P F P F P F P F P F P

N 71.84 <0.0001 60.07 <0.0001 7.41 0.01 16.22 0.0003 0.02 0.89 10.83 0.002P 3.45 0.06 0.11 0.74 4.32 0.04 0.30 0.59 0.06 0.81 0.21 0.65Litter 314 <0.0001 0.03 0.86 75.70 <0.0001 0.09 0.77 9.25 0.004 0.05 0.82Litter � N 2.53 0.11 1.04 0.31 8.50 0.006 0.70 0.41 0.02 0.88 0.01 0.93Litter � P 2.86 0.09 1.19 0.28 3.23 0.08 0.24 0.63 1.10 0.30 0.82 0.37

M. Fisk et al. / Soil Biology & Biochemistry xxx (2014) 1e74

1234567891011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556575859606162636465

6667686970717273747576777879808182838485868788899091929394959697

SBB6047_proof ■ 1 December 2014 ■ 4/7

litter suppressed C mineralization immediately relative to addedlitter alone. By the end of the incubation, C mineralized from thelitter þ N treatment was comparable to that from untreated con-trols (Fig. 2A). Although we cannot conclusively distinguish be-tween mineralization of litter C vs SOC in litter treatments, thesubstantial effect of added N on C mineralization rates in theabsence of litter suggest a strong effect on SOC. Subtracting total Cmineralized in controls from that in treatment incubations showsthe net suppression by N, which is only slightly larger (13%) whenlitter is added compared to soils with no added litter (Fig. 3).

In contrast to the uniformly negative effects of N, effects ofadded P depended on the litter treatment (Table 2). The overalleffect of added P was marginally significant as was the litter � Pinteraction (Table 2). The P treatment alone did not affect miner-alization of SOC at any time during the incubation (Fig. 2B),whereas adding litter þ P significantly increased C mineralizationrates relative to litter alone (t ¼ �2.51, p ¼ 0.06). The litter þ Ptreatment did not alter respiration in the first 2 d; however, be-tween days 4 and 8 rates increased relative to the litter-onlytreatment (Fig. 2B). As a consequence, P increased the total litter-

Fig. 2. Rates of C mineralization from untreated controls and from treatmentsreceiving (A) leaf litter (Le) and N and (B) Le and P. Data points are means of 9 stands.See Table 2 for statistical comparisons.

9899

100101102103104105106107

Please cite this article in press as: Fisk, M., et al., Carbon mineralizationorganic horizon of northern hardwood forests, Soil Biology & Biochemist

related C mineralization by approximately 30% (Fig. 3). Forest agedid not influence nutrient effects, with or without added litter.

Added glucose-C was mineralized rapidly in incubations from asmall subset of our forest stands (Fig. 4A), exceeding that miner-alized from controls (400e485 mg C g�1 d�1; data not shown) by4e5 times during the first 2 days. A total of 25e30 % of addedglucose C was mineralized in about 4 days, after which C miner-alization rates in the glucose treatment were equal to or less thancontrols. Nitrogen and P effects were not consistent between standsand had no significant effects on C mineralization (Table 3).

Carbon was mineralized from added cellulose relatively slowly,totaling only 9.1% of added C at the end of the incubation (Fig. 4B).In combination with cellulose, nutrient additions elicited similarresponses to those with added leaf litter, with significant effects ofN and P (Fig. 4B, Table 3). Added N appeared to suppress mineral-ization of C from SOC aswell as from added cellulose, reducing ratesin the cellulose þ N treatment below those of controls such thattotal C mineralized was only 80% of that from control soils (Fig. 2). Atotal of 26% of added cellulose-C was mineralized in the P treat-ment, more than twice what was mineralized in the cellulose-onlytreatment (Fig. 2).

3.3. Treatment responses: inorganic N and microbial biomass

Adding leaf litter reduced net N mineralization from an averageof 4.8 mg N g�1 d�1 in control soils to 0.7 mg N g�1 d�1 in litter-amended soils. At the end of the incubation, inorganic N in thelitter treatment was less than half that of controls (Table 4). Addingleaf litter had no detectable effect on MBN or DON, whereas MBNand DON pools increased significantly in response to N addition

Fig. 3. Differences in total C mineralization between treatments receiving addedorganic matter (OM; litter, cellulose, glucose) and N or P, and control (untreated) soils.No external OM was added to the SOM treatment. Error bars are standard errors of themean; n ¼ 9 stands for SOM and litter and n ¼ 2 for cellulose and glucose.

108109110111112113114115116117118119120121122123124125126127128129130

is promoted by phosphorus and reduced by nitrogen addition in thery (2014), http://dx.doi.org/10.1016/j.soilbio.2014.11.022

Fig. 4. Rates of C mineralization from treatments receiving (A) glucose, glucose þ N,and glucose þ P, and (B) cellulose, cellulose þ N, and cellulose þ P. Data points are theaverage of soil from one mature and one mid-age plot. For statistical differences, seeTable 3.

Table 3Anova results (F ratios and P values) for the repeated measures model testing the Csource � nutrient addition interaction effects on C mineralization.

Source Glucose Cellulose

F P F P

N 0.00 0.98 8.59 0.005P 0.04 0.85 5.53 0.022

M. Fisk et al. / Soil Biology & Biochemistry xxx (2014) 1e7 5

1234567891011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556575859606162636465

66676869707172737475767778798081828384858687888990919293949596979899

100101102103104105106107108109110111112113114115116117

SBB6047_proof ■ 1 December 2014 ■ 5/7

(Table 2; Table 4). Effects of litterþNwere similar to those of addedN alone; there was no interaction of litter � N. Added P had nodetectable effects on MBN (Table 2).

We estimated the amount of added N that ended up in theinorganic N, MBN, and DON pools as the difference between the Ntreatment and controls (Table 4), and were able to account forabout 80% of added N in these three pools. The accumulation of N inthe inorganic pool equaled about 37% of added N, compared to 21%in MBN and 24% in DON (Table 4).

Leaf litter addition increased extractable DOC, an effect thatincreased in the presence of added N or P. While N and P effects onDOC were significant, DOC did not differ from controls when eithernutrient was added without litter, causing significant litter �nutrient interactions (Table 2). Microbial biomass C responded toadded litter but not to nutrients (Table 2), increasing by 12e15% inlitter treatments (Table 4). Microbial C:N responded only to N(Table 2), averaging 10.5 in controls compared to 7.4 in the Ntreatment.

118119120121122123124125126127128129130

4. Discussion

In this study, we found that N suppresses and P enhancesmineralization of C in the surface organic horizon in northernhardwood forests. Phosphorus did not affect mineralization ofnative SOC, but consistently across sites increased that of C added aslitter and cellulose, whereas N substantially reducedmineralizationof SOC aswell as that of C added as litter and cellulose (Fig. 3). Theseresults suggest that P promotes respiratory losses related to new Cinputs whereas available N reduces C emission from the organichorizon in these forests, summarized in Fig. 5. While the strength ofthese mechanisms varied across our sites, we found consistency in

Please cite this article in press as: Fisk, M., et al., Carbon mineralizationorganic horizon of northern hardwood forests, Soil Biology & Biochemist

the direction of effects across sites and between successional andmature forests.

We also found that N increased MBN, indicating N limitation ofmicrobial N assimilation and agreeingwith expectations for organichorizons with high C:N (Schimel and Weintraub, 2003; Lee andSchmidt, 2014). The increase in extractable DON after N additionsuggests further microbial assimilation, followed by turnover toproduce soluble N products (Schmidt et al., 2007). Higher proteincontent ofmicrobial biomass could contribute to lower C:N, and it isalso possible that extracellular enzyme production increases withavailableN (Ratliff, 2012). Substantial turnoverofN throughbiomassinto stabilized forms is likely as well, given the relatively largeamount of added N that remained unaccounted for in inorganic,dissolved, or microbial biomass pools (Table 4). The reduction ofmicrobial biomassC:N in response toNcontrastswith conclusionsofMooshamer et al. (2014) that adjustments in nutrient use effi-ciencies maintain microbial stoichiometry, and suggests that suchadjustments may be sensitive to initial nutrient limitation.

The strong suppression of C mineralization by N in our studyagrees with findings of some studies (Fontaine et al., 2004; Craineet al., 2007; Bradford et al., 2008; Fanin et al., 2012; Ramirez et al.,2012), but not others (Allen and Schlesinger, 2004; Milcu et al.,2011; Reed et al., 2011). In our study the primary effect of Nappeared to be on mineralization of SOC. We estimated litter-related C mineralization by subtracting control rates. The N effecton Cmineralizationwas larger for litter-related C compared to SOM(Fig. 3), suggesting some suppression by N of mineralization oflitter C. However, we cannot with our methods separate priming ofSOC from mineralization of litter-derived C.

While it is clear that added N reduced C emissions from the Oehorizon organic matter, the fate of the C not respired is less obvious.Nitrogen reduced C mineralization by an average of 2110 mg C/g soilover the course of the incubation, an amount equal to approximately25%of themicrobial Cpool.Maintaining the samemicrobial biomassC pools with less microbial respiration (C mineralization) in þNcompared to control soils requires either slower turnover of themicrobial C pool or higher microbial growth efficiency (the fractionof total C uptake that is assimilated for biomass synthesis). If N re-duces turnover through the microbial biomass C pool in addition toreducing respiration, then biomass C could be maintained at a levelsimilar to controls without any change in microbial growth effi-ciency. In contrast, if N does not affect turnover then the amount of Cassimilated for biomass synthesis would also be unaffected by N.Compared to controls, similar C assimilation but lower respirationwould mean higher microbial growth efficiency in response to N. Ifbiomass turnover increased in response to N, then a larger increasein microbial growth efficiency would be required to maintainbiomass levels comparable to controls. Without knowing microbialgrowth and turnover rates it is difficult to conclusively interpret thefate of C that was not respired in the þN treatment relative to con-trols. Better estimates of the turnover of C through the microbialpool are needed to learn whether decomposition was slowed byadded N, reducing the uptake and processing of C by the microbialbiomass, or whether microbial biomass grew more efficiently inresponse to N, transforming more C into other forms of soil organicmatter.

is promoted by phosphorus and reduced by nitrogen addition in thery (2014), http://dx.doi.org/10.1016/j.soilbio.2014.11.022

Table 4Nitrogen in organic and inorganic pools, and C inmicrobial biomass, averaged across all sites and both forest ages (n¼ 9). Initial inorganic N concentrationwas 40 mg/g soil, plus2000 mg/g soil in þN treatments. Positive values of (treatment-control) indicate accumulation in a pool.

Final values after incubation (mg/g) Inorganic N Treatment e control (mg/g)

Inorganic N DON MBN MBC DON MBN MBC

Control 137 (27) 115 (14) 797 (95) 8518 (813)þLitter 57 (7) 114 (25) 801 (108) 9594 (534) �89 (23) 4 (23) 16 (33) 1076 (963)þN 959 (94) 492 (100) 1089 (166) 8660 (608) 810 (77) 414 (109) 491 (109) 142 (641)þLitter þ N 848 (81) 616 (101) 1075 (50) 9590 (633) 699 (65) 555 (118) 347 (146) 1072 (534)þP 149 (19) 161 (36) 640 (107) 7834 (497) 0 (14) 47 (40) �99 (116) �527 (807)þLitter þ P 42 (12) 40 (15) 822 (50) 10059 (587) �106 (30) �72 (20) 54 (103) 1323 (611)

M. Fisk et al. / Soil Biology & Biochemistry xxx (2014) 1e76

1234567891011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556575859606162636465

66676869707172737475767778798081828384858687888990919293949596979899

100

SBB6047_proof ■ 1 December 2014 ■ 6/7

In contrast to the suppressive effects of N, P addition in com-bination with C enhanced microbial respiration. Phosphorus limi-tation of microbial respiratory activity in forest floor of these northtemperate forests contributes to growing recognition of theimportance of P availability to the microbial processes that influ-ence soil C balance (Cleveland et al., 2006; Craine et al., 2007;Bradford et al., 2008; Hartman and Richardson, 2013). Phos-phorus effects can be especially pronounced in ecosystemswhere Plimits aboveground processes (Reed et al., 2011). While N may beexpected to limit productivity of northern hardwood forests, evi-dence does exist for P limitation and co-limitation (Vadeboncoeur,2010). In some of our sites at the BEF, root foraging suggests highdemand for P, especially those that are mid-aged (Naples and Fisk,2010), and modeling suggests that nutrient limitation of forestproductivity will shift between P, N, and co-limitation over suc-cessional time (Rastetter et al., 2013). However, we found no forestage effects on respiration or response to nutrients. In fact theconsistency in direction of P effect across forest age and sites issomewhat surprising, given the wide variation in N and P avail-ability across sites (Ratliff, 2012; Fisk et al., 2014).

Effects of P on C mineralization from litter, and not from SOC,suggest that organic C inputs are required to induce secondarylimitation by P in these forests. In the northern hardwoods, if the

Fig. 5. Movement of C into and out of microbial biomass (shaded). Line thicknessrepresents relative differences in C movement related to addition of N and addition ofleaf litter þ P. Black lines indicate variables that were measured and blue linesrepresent hypothesized responses to treatments. (For interpretation of the referencesto color in this figure legend, the reader is referred to the web version of this article.)

101102103104105106107108109110111112113114115116117118119120121122123124125126127128129130

Please cite this article in press as: Fisk, M., et al., Carbon mineralizationorganic horizon of northern hardwood forests, Soil Biology & Biochemist

secondary limitation by P arises in response to microbial growth,then the response found here is consistent with the Growth RateHypothesis, in which more rapid growth requires more P for ribo-somes to encode proteins (Elser et al., 1996). The dependence onadded C is similar to findings by Reed et al. (2011) in tropical forests,and is consistent with results of another study at HBEF, in whichadded P had no effect on SOC mineralization (Groffman and Fisk,2011; Minick et al., 2011). However, it contrasts with findings ofother studies in which P enhanced mineralization from SOC as wellas from added C (Cleveland et al., 2006; Bradford et al., 2008).

Interactions between N and C availability deserve attention forunderstanding why the response to P requires added C in somesystems but not others. A primary constraint on growth by theavailability of C could seem contradictory in the organic-rich Oehorizon (about 80% OM). However, it would make sense if theavailability of small C molecules is itself dependent on the avail-ability of N to construct extracellular enzymes needed to breakdown plant structural material (Schimel and Weintraub, 2003).Thus, our P and N responses agree with a framework in which Nlimits the availability of C for uptake by microorganisms in thisorganic rich horizon, and we summarize these hypothesized effectsin Fig. 5. A factorial study of the effects of N þ P together supportsthis idea in tropical forests on young soils (Reed et al., 2011), and isneeded to examine this possibility in temperate forests, especiallythosewith thick surface organic horizons. However, this interactioncould be difficult to detect in our soils given the opposing effects ofN and P on C mineralization, highlighting the need to distinguishbetween microbial growth and respiratory processes (Lee andSchmidt, 2014).

It is not obvious why both N and P would alter respiratory re-sponses to added litter and added cellulose, but not to addedglucose. This lack of consistency among substrate types emphasizesthe possibility of different resource demands associated with thecomplexity of the breakdown process. In this case metabolism ofsimple C compounds could be less limited by the availability of N iffewer proteins are required, compared to metabolism of structuralC. The contrast between C sources could also correspond withdifferential responses within the microbial community, because ofdifferences in enzymatic pathways or other traits. For example,glucose could be rapidly metabolized by a fast-growing subset ofthe microbial community with inherently different growth effi-ciency compared to the remainder of the community. Growth ef-ficiency of individual taxa has been shown to vary in response toresource stoichiometry, and bacterial and fungal responses areespecially likely to differ (Meidute et al., 2008; Keiblinger et al.,2010). The contrast between glucose and cellulose effects, and thelikely importance of oligotrophic and copiotrophic components ofmicrobial communities (Fierer et al., 2012), suggest the importanceof the nature of C sources that drive microbial activity.

In summary, high P availability promoted respiratory lossesfrom added litter and high N availability reduced respiration fromSOC and litter (Fig. 5), but promoted N movement into microbialbiomass and DON and reduced microbial C:N. These results were

is promoted by phosphorus and reduced by nitrogen addition in thery (2014), http://dx.doi.org/10.1016/j.soilbio.2014.11.022

Q7

Q4

Q5

M. Fisk et al. / Soil Biology & Biochemistry xxx (2014) 1e7 7

1234567891011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556575859606162636465

6667686970717273

SBB6047_proof ■ 1 December 2014 ■ 7/7

consistent across several northern hardwood forest ecosystems andboth mid-successional and mature forests. Our results support theconclusion by Hartman and Richardson (2013) that N limitsbiomass synthesis and P limits metabolism, if we assume that Naddition did not reduce C turnover through the microbial pool. Ourresults emphasize the need to better understand turnover andenzyme production in order to elucidate nutrient effects on thetransformations of soil organic C.

7475767778

Uncited reference

Keuskamp et al., 2013

79808182838485868788899091

Acknowledgments

We thank Tera Ratliff and Matt Negrey for assistance in the fieldand laboratory. This work was supported by a grant from the Na-tional Science Foundation and by an REU supplement to that grant.This manuscript is a contribution of the Hubbard Brook EcosystemStudy. Hubbard Brook is part of the Long-Term Ecological Research(LTER) network, which is supported by the National ScienceFoundation. The Hubbard Brook Experimental Forest is operatedand maintained by the USDA Forest Service, Northern ResearchStation, Newtown Square, PA, USA.

9293949596979899

100101102103104105106107108109110111112113114115116117118119120121122123124125126127128129130

References

Aber, J.D., Goodale, C.L., Ollinger, S.V., Smith, M., Magill, A.H., Martin, M.E.,Hallett, R.A., Stoddard, J.L., 2003. Is nitrogen deposition altering the nitrogenstatus of Northeastern forests? Bioscience 53, 375e389.

Allen, A.S., Schlesinger, W.H., 2004. Nutrient limitations to soil microbial biomassand activity in loblolly pine forests. Soil Biology and Biochemistry 36,581e589.

Bradford, M.A., Fierer, N., Reynolds, J.F., 2008. Soil carbon stocks in experimentalmesocosms are dependent on the rate of labile carbon, nitrogen, and phos-phorus inputs to soils. Functional Ecology 22, 964e974.

Bohlen, P.J., Groffman, P.M., Driscoll, C.T., Fahey, T.J., Siccama, T.G., 2001. Plant-soil-microbial interactions in a northern hardwood forest. Ecology 82,965e978.

Brookes, P.C., Landman, A., Pruden, G., Jenkinson, D.S., 1985. Chloroform fumigationand the release of soil nitrogen: a rapid direct extraction method for measuringmicrobial biomass nitrogen in soil. Soil Biology and Biochemistry 17, 837e842.

Carreira, J.A., Garcia-Ruiz, R., Lietor, J., Harrison, A.F., 2000. Changes in soil phos-phatase activity and P transformation rates induced by application of N- and S-containing acid mist to a forest canopy. Soil Biology and Biochemistry 32,1857e1865.

Cleveland, C.C., Townsend, A.R., Schmidt, S.K., 2002. Phosphorus limitation of mi-crobial processes in moist tropical forests: evidence from short-term laboratoryincubations and field studies. Ecosystems 5, 680e691.

Cleveland, C.C., Townsend, A.R., 2006. Nutrient additions to a tropical rain forestdrive substantial soil carbon dioxide losses to the atmosphere. Proceedings ofthe National Academy of Science 103, 10316e10321.

Craine, J.M., Morrow, C., Fierer, N., 2007. Microbial nitrogen limitation increasesdecomposition. Ecology 88, 2105e2113.

Dittman, J.A., Driscoll, C.T., Groffman, P.M., Fahey, T.J., 2007. Dynamics of nitrogenand dissolved organic carbon at the Hubbard Brook experimental Forest.Ecology 88, 1153e1166.

Elser, J.J., Dobberfuhl, D.R., MacKay, N.A., Schampel, J.H., 1996. Organism size, lifehistory, and N: P stoichiometry. BioScience 46, 674e684.

Fanin, N., Barantal, S., Fromin, N., Schimann, H., Schevin, P., Hattenschwiler, S., 2012.Distinct microbial limitations in litter and underlying soil revealed by carbonand nutrient fertilization in a tropical rainforest. PLoS One 7, 1e11.

Fierer, N., Lauber, C.L., Ramirez, K.S., Zaneveld, J., Bradford, M.A., Knight, R., 2012.Comparative metagenomic, phylogenetic, and physiological analyses of soilmicrobial communities across nitrogen gradients. ISME Journal 6, 1007e1017.

Fenn, M.E., Poth, M.A., Aber, J.D., Baron, J.S., Bormann, B.T., Johnson, D.W.,Lemly, A.D., McNulty, S.G., Ryan, D.F., Stottlemeyer, R., 1998. Nitrogen excess inNorth American ecosystems: a review of predisposing factors, geographicextent, ecosystem responses and management strategies. Ecological Applica-tions 8, 706e733.

Fiorentino, I., Fahey, T.J., Groffman, P.M., Driscoll, C.T., Eager, C., Siccama, T.G., 2003.Initial response of phosphorous biogeochemistry to calcium addition in anorthern hardwood forest ecosystem. Canadian Journal of Forest Research 33,1864e1873.

Please cite this article in press as: Fisk, M., et al., Carbon mineralizationorganic horizon of northern hardwood forests, Soil Biology & Biochemist

Fisk, M.C., Fahey, T.J., 2001. Microbial biomass and nitrogen cycling response tofertilization and litter removal in young northern hardwood forests. Biogeo-chemistry 53, 201e223.

Fisk, M.C., Zak, D.R., Crow, T.R., 2002. Nitrogen cycling and retention in old- andsecond-growth northern hardwood forests. Ecology 83, 73e87.

Fisk, M.C., Fahey, T.J., Groffman, P.M., 2010. Carbon resources, soil organisms, andnitrogen availability: landscape patterns in a northern hardwood forest. ForestEcology and Management 260, 1175e1183.

Fisk, M.C., Ratliff, T.J., Goswami, S., Yanai, R.D., 2014. Synergistic soil response tonitrogen plus phosphorus fertilization in hardwood forests. Biogeochemistry118, 195e204.

Fontaine, S., Bardoux, G., Abbadie, L., Mariotii, A., 2004. Carbon input to soil maydecrease soil carbon content. Ecology Letters 7, 314e320.

Frey, S.D., Six, J., Elliot, E.T., 2003. Reciprocal transfer of carbon and nitrogen bydecomposer fungi at the soil-litter interface. Soil Biology and Biochemistry 35,1001e1004.

Groffman, P.M., Fisk, M.C., 2011. Phosphate additions have no effect on microbialbiomass and activity in a northern hardwood forest. Soil Biology andBiochemistry 44, 2441e2449.

Hartman, W.H., Richardson, C.J., 2013. Differential nutrient limitation of soil mi-crobial biomass and metabolic quotients (qCO2): is there a biological stoichi-ometry of soil microbes? PLoS One 8, 1e14.

Johnson, C.E., Driscoll, C.T., Siccama, T.G., Likens, G.E., 2000. Element fluxes andlandscape position in a northern hardwood forest watershed ecosystem. Eco-systems 3, 159e184.

Kamble, P.N., Bååth, E., 2014. Induced N-limitation of bacterial growth in soil: effectof carbon loading and N status in soil. Soil Biology and Biochemistry 74, 11e20.

Keuskamp, J.A., Schmitt, H., Laanbroek, H.J., Verhoeven, J.T.A., Hefting, M.M., 2013.Nutrient amendment does not increase mineralization of sequestered carbonduring incubation of a nitrogen limited mangrove soil. Soil Biology andBiochemistry 57, 822e829.

Keiblinger, K.M., Hall, E.K., Wanek, W., Szukics, U., H€ammerle, I., Ellersdorfer, G.,B€ock, S., Strauss, J., Sterflinger, K., Richter, A., Zechmeister-Boltenstern, S., 2010.The effect of resource quantity and resource stoichiometry on microbial carbon-use-efficiency. FEMS Microbiology Ecology 73, 430e440.

Lawrence, G.B., David, M.B., Shortle, W.C., 1995. A new mechanism for calcium lossin forest-floor soils. Nature 378, 162e165.

Lee, Z.M., Schmidt, T.M., 2014. Bacterial growth efficiency varies in soils underdifferent land management practices. Soil Biology and Biochemistry 69,282e290.

Likens, G.E., Driscoll, C.T., Buso, D.C., Siccama, T.G., Johnson, C.E., Lovett, G.M.,Fahey, T.J., Reiners, W.A., Ryan, D.F., Martin, C.W., Bailey, S.W., 1998. Thebiogeochemistry of calcium at Hubbard Brook. Biogeochemistry 41, 89e173.

McLauchlan, K.K., Craine, J.M., Oswald, W.W., Leavitt, P.R., Likens, G.E., 2007.Changes in nitrogen cycling during the past century in a northern hardwoodforest. PNAS 104, 7466e7470.

Meidute, S., Demoling, F., Bååth, E., 2008. Antagonistic and synergistic effects offungal and bacterial growth in soil after adding different carbon and nitrogensources. Soil Biology and Biochemistry 40, 2334e2343.

Milcu, A., Heim, A., Ellis, R.J., Scheu, S., Manning, P., 2011. Identification of generalpatterns of nutrient and labile carbon control on soil carbon dynamics across asuccessional gradient. Ecosystems 13, 710e719.

Minick, K.J., Fisk, M.C., Groffman, P.M., 2011. Calcium and phosphorus interact toreduce mid-growing season net nitrogen mineralization potential in organichorizons in a northern hardwood forest. Soil Biology and Biochemistry 43,271e279.

Mooshamer, M., Wanek, W., Zechmeister-Boltenstern, S., Richter, A., 2014. Stoi-chiometric imbalances between terrestrial decomposer communities and theirresources: mechanisms and implications of microbial adaptations to their re-sources. Frontiers in Microbiology 5, 1e10.

Naples, B.K., Fisk, M.C., 2010. Belowground insights into nutrient limitation innorthern hardwood forests. Biogeochemistry 97, 109e121.

Ramirez, K.S., Craine, J.M., Fierer, N., 2012. Consistent effects of nitrogen amend-ments on soil microbial communities and processes across biomes. GlobalChange Biology 18, 1918e1927.

Rastetter, E.B., Yanai, R.D., Thomas, R.Q., Vadeboncoeur, M.A., Fahey, T.J., Fisk, M.C.,Kwiatkowski, B.L., Hamburg, S.P., 2013. Recovery from disturbance requiresresynchronization of ecosystem nutrient cycles. Ecological Applications 23,621e642.

Ratliff, T.J., 2012. Nitrogen and Phosphorus Availability in Forests of Varying Ages inthe White Mountains, New Hampshire (MS thesis). Miami University.

Reed, S.C., Vitousek, P.M., Cleveland, C.C., 2011. Are patterns in nutrient limitationbelowground consistent with those aboveground: results from a 4 million yearchronosequence. Biogeochemistry 106, 323e336.

Schimel, J.P., Weintraub, M.N., 2003. The implications of exoenzyme activity onmicrobial carbon and nitrogen limitation in soil: a theoretical model. SoilBiology and Biochemistry 35, 549e563.

Schmidt, S.K., Costello, E.K., Nemergut, D.R., Cleveland, C.C., Reed, S.C.,Weintraub, M.N., Meyer, A.F., Martin, A.M., 2007. Biogeochemical consequences ofrapid microbial turnover and seasonal succession in soil. Ecology 88, 1379e1385.

Vadeboncoeur, M.A.V., 2010. Meta-analysis of fertilization experiments indicatesmultiple limiting nutrients in northeastern deciduous forests. Canadian Journalof Forest Research 40, 1766e1780.

Vance, E.D., Brookes, P.C., Jenkinson, D.S., 1987. An extraction method for measuringsoil microbial biomass C. Soil Biology and Biochemistry 19, 703e708.

is promoted by phosphorus and reduced by nitrogen addition in thery (2014), http://dx.doi.org/10.1016/j.soilbio.2014.11.022