Geomicrobiology Journal, 23:531–543, 2006Copyright c© Taylor & Francis Group, LLCISSN: 0149-0451 print / 1521-0529 onlineDOI: 10.1080/01490450600897278

Nutrient Input and Carbon and Microbial Dynamicsin an Ombrotrophic Bog

Nathan Basiliko and Tim R. MooreDepartment of Geography and Centre for Climate & Global Change Research, McGill University,Sherbrooke St. West Montreal, Quebec, Canada H3A 2K6

Richard JeannotteDepartment of Natural Resource Sciences, McGill University, Macdonald Campus, St. Anne de Bellevue,QC, Canada H9X 3V9

Jill L. BubierEnvironmental Studies Program, Department of Earth and Environment, Mount Holyoke College, SouthHadley, Massachusetts, USA 01075

Slow rates of plant production and decomposition in om-brotrophic bogs are believed to be partially the result of low nu-trient availability. To test the effect of nutrient availability ondecomposition, carbon dioxide (CO2) flux dynamics, microbialbiomass, and nutrients, we added nitrogen (N) with phosphorus(P) and potassium (K), to prevent limitation of the latter 2 nu-trients, over 2 growing seasons to plots at Mer Bleue peatland,Ontario, Canada. After the first growing season, increasing N fer-tilization (with constant P and K) decreased in vitro CO2 productionpotential and increased microbial biomass measured with a chlo-roform fumigation-extraction technique in the upper peat profile,while by the end of the second season, CO2 production potentialwas increased in response to N plus PK treatment, presumably dueto more easily decomposable newly formed plant material. In situCO2 fluxes measured using chamber-techniques over the second

Received 30 January 2006; accepted 28 March 2006.Support came from the Natural Sciences and Engineering Research

Council of Canada to TRM. NB was supported by graduate fellow-ships from McGill University and the Centre for Climate and GlobalChange Research. JB was supported by a grant from NASA’s EarthSystem Science Program. We thank Henri Dinel and Louise Florent(Agriculture Canada, ECORC) for use of and assistance with the sol-vent extractor, Joe Yavitt, Kurt Smemo (Department of Natural Re-sources, Cornell University) and Mike Rutske (Department of PlantBiology, Cornell University) for ICP analysis, Mike Dalva (Depart-ment of Geography, McGill University) and Helene Lalande (Depart-ment of Natural Resource Sciences, McGill University) for providingexcellent technical assistance, Emily Neal, Gaytri Bhatia, and MeaghanMurphy (Mount Holyoke College) for field and laboratory assistance,and Leszek Bledzki (Mount Holyoke College) for statistical analyses.We also thank two anonymous reviewers for helpful suggestions onrevising an earlier draft of the manuscript.

Address correspondence to Nathan Basiliko, Department of ForestSciences, University of British Columbia, 2424 Main Mall Vancouver,British Columbia, Canada V6T 1Z4. E mail: nathan.basiliko@ubc.ca

year corroborated this presumption, with greater photosyntheticCO2 uptake and ecosystem respiration (ER) during high N plus PKtreatments. The more efficient microbial community, with slowerCO2 production potential and larger biomass, after the first yearwas characterized by larger fungal biomass measured with signa-ture phospholipid fatty acids. The majority of N was likely quicklysequestered by the vegetation and transferred to dissolved organicforms and microbial biomass in the upper parts of the peat profile,while additional P relative to controls was distributed throughoutthe profile, implying that the vegetation at the site was N limited.However, in situ CO2 flux data suggested the possibility of P orNPK limitation. We hypothesize that nutrient deposition may leadto enhanced C uptake by altering the microbial community and de-composition, however this pattern disappears through subsequentchanges in the vegetation and production of more readily decom-posable plant tissues.

Keywords carbon dioxide, microbial biomass, nitrogen, PLFA, peat-land, phosphorus

INTRODUCTIONNorthern peatlands are important long-term sinks for atmo-

spheric carbon dioxide (CO2), resulting from net primary pro-duction (NPP) exceeding organic matter decomposition. Peat-lands have stored between 270 and 455 Gt of CO2-C over theHolocene, with long-term accumulation rates of between 15and 30 g C m−2 yr−1 (Gorham 1991; Turunen et al. 2002). In-creased nutrient, especially nitrogen (N), deposition from indus-trial pollution has the potential to drastically alter the biogeo-chemistry, microbiology, and carbon (C) balance of terrestrialecosystems (Matson et al. 2002). The net effects of increased de-position are complex, because nutrient additions can potentiallyaffect both NPP and organic matter decomposition. It is unclearwhat role increased nutrient deposition plays in peatland Cbiogeochemistry.

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532 N. BASILIKO ET AL.

Vegetation in these primarily rain-fed, ombrotrophic north-ern peatlands is N-limited in unpolluted regions of NorthAmerica and N is largely sequestered by the moss layer andretained by Sphagnum mosses and the upper peat profile (Liand Vitt 1997). Nitrogen saturation may occur in polluted re-gions, leading to increased N mineralization rates (Lamers et al.2000). Critical nitrate (NO3)-N deposition values above whichenhanced NPP ceases and stress occurs in many Sphagnummosses, have been estimated to be 1.5 g N m−2 yr−1 (Vittet al. 2003). Nitrogen fertilization has been shown to both en-hance and decrease NPP, depending on rates of fertilization andambient deposition, on species of Sphagnum (Jauhiainen et al.1998), and on climate and environmental conditions (Aerts et al.2001).

The slow rates of peat decomposition in northern peatlandsresult from cold temperatures, anaerobic conditions (Blodauet al. 2004), anti-microbial properties of Sphagnum mosses(Verhoeven and Liefveld 1997; Yavitt et al. 2000), low pH(Williams et al. 2000) and low nutrient availability in ombroge-nous bog sites. Nitrogen fertilization has been shown to en-hance (Coulson and Butterfield 1978), have no effect (Rochefortet al.1990; Aerts et al. 2001; Tommasen et al. 2004), or decrease(Williams and Silcock 1997) peat decomposition rates; howeverdifferent methods were used in these studies to investigate peatdecomposition, making comparisons between them difficult. In-organic N addition to soil organic matter has been often shownto retard decomposition, particularly with organic matter that isrecalcitrant (Fog 1988; Neff et al. 2002). Agren et al. (2001)proposed three mechanisms to explain this effect: decreaseddecomposer growth rate, altered microbial community withincreased decomposer efficiency (CO2 production to biomassassimilation ratio), and more rapid formation of recalcitrantcompounds.

The objective of this study was to characterize the effects of Nfertilization on peat decomposition, CO2 exchange, and nutrientand microbial biomass dynamics at Mer Bleue bog near Ottawa,Canada. The hypothesis that decreased decomposition resultsfrom inorganic N additions (Williams and Silcock 1997) wastested. Field additions of N, with phosphorus (P) and potassium(K) to prevent limitation of these nutrients, were applied to bogplots over two years and peat and microbial characteristics suchas biomass and phospholipid fatty acids (PLFAs), as well as theCO2 production potential of the upper part of the peat profile, andin situ CO2 flux dynamics were measured. The field experimentallowed an evaluation of the effect of vegetation in mediatingresponse to nutrient additions.

METHODS

Study SiteThe Mer Bleue bog, located 10 km east of Ottawa, Canada, is a

large peatland complex with hummock-hollow patterned topog-raphy. The dominating hummocks contained Sphagnum capil-lifolium, Sphagnum magellanicum, and Polytrichum strictum

mosses and Chamaedaphne calyculata, Ledum groenlandicumand Kalmia angustifolia shrubs. Hollows primarily contained S.magellanicum and Sphagnum angustifolium. Surface elevationbetween hummocks and hollows varied by 10 to 20 cm. The wa-ter table depth over the year 2000 growing season ranged from10 to 50 cm beneath the surface of the hummocks. The late sum-mer in 2001 was considerably drier, with water tables droppingbelow 50 cm. Total N deposition rate from a Wet atmosphericN deposition was estimated from Keith and Dillon (1989) andSisterson et al. (1994) to be 0.6 to 0.8 g N m2 yr−1.

Eddy covariance tower measurements of CO2 exchangeshowed that the bog was, on average a net C sink ranging from−10 to 100, average 30 g C m−2 yr−1 over 6 years, after account-ing for C losses as methane (CH4)-C efflux and dissolved organicand inorganic C (DOC and DIC) export (Lafleur et al. 2003; pers.comm.). This inter-annual variability in C exchange, comparedto long-term C accumulation rates of 15 to 20 g C m−2 yr−1,arises from differences in photosynthetic and respiration ratesassociated with variations in weather.

Field FertilizationFertilization plots (2.25 or 9 m2 in area) were set up in a 400

m2 section of the bog with little variation in micro-topographyand vegetation. Solutions of N were added bi-weekly as ammo-nium nitrate (NH4NO3) at 3.2 and 6.4 g N m−2 yr−1, approxi-mately 10 and 20 times the ambient growing season atmosphericdeposition. Phosphorus and K were added as potassium dihy-drogen phosphate (KH2PO4), equivalent to 6.3 g K and 5.0 g Pm−2 yr−1. Control plots received P and K solution or distilledH2O. Triplicate plots for field flux measurements were identifiedas 0NPK, PK, 10N+PK, and 20N+PK. The fertilization plotswere established as part of a longer-term study to investigateeffects of chronic nutrient additions. Therefore we had to limitdestructive sampling for laboratory analysis to one plot for eachtreatment. Because our treatment plots were large, in a homoge-nous area of vegetation and topography, near each other, andbecause we added large concentrations of nutrients, we assumedthat observed differences were due to treatments. We must ac-knowledge, however, that replicate samples analyzed in the labwere not taken from individual, randomly assigned treatmentplots.

Two weeks after the final fertilization in September 2000,sets of five 10 cm2 by 35 cm deep cores were removed with aserrated knife from single 0NPK, 10N+PK, and 20N+PK plotsand transported in coolers to McGill University. The bottom ofthe cores was approximately at the transition from unsaturatedto saturated peat at the time of sampling. Vegetation, includ-ing photosynthetic portions of surface Sphagnum moss stemswere removed by cutting and cores were divided into 0–5, 5–10, 10–20, and 20–30 cm depth segments prior to determina-tion of CO2 production potential, microbial biomass C and N,extractable organic C and N, inorganic N, pH, and elementalanalysis. Microbial PLFAs were characterized in the 0–5 cm

NUTRIENT EFFECTS IN AN OMBROTROPHIC BOG 533

depth segments from the 0NPK and 20N+PK plots. Two weeksafter the final fertilization in September 2001, sets of five coreswere removed from the same 0NPK and 20N+PK plots as aboveand a single PK plot and segments were divided into two equalparts. Roots were left intact in one and were removed in theother, divided into coarse and fine categories (diameter >2 and<2 mm diameter, respectively), and dried and weighed to cal-culate root biomass. Respiration potential, microbial biomassC, N and P, extractable organic C, N, and P, pH, and inorganicN and P were determined for sub-samples with and withoutroots.

Analytical Methods and CalculationsCO2 production potential under aerobic conditions was de-

termined by placing ca. 5 g of peat (wet weight) from eachsample into a 50-ml Erlenmeyer flask, while taking care towash utensils and gloved hands before handling each sample.Twenty ml of deionized water was added to create a slurrythat helped to eliminate any local regions of anoxia in thepeat. Flasks were sealed with silicon filled rubber Suba seals(William Freeman Ltd, Barnsley, UK) and shaken on a rotaryshaker at 200 rpm for 2 days. After the incubation, the pH of theslurry was measured potentiometrically (Watertest AgS/glass-electrode, Hanna Instruments, Woonsocket, RI, USA) with anOrion meter (Thermo Electron Corp., Waltham, MA, USA), theheadspace volume was determined by displacement with wa-ter, and the content in each flask was dried at 70◦C to mea-sure the dry weight of peat. During the incubation, gas fromthe headspace was sampled at least 3 times and analyzed forrate calculations. Carbon dioxide concentrations in headspaceair were measured using a Shimadzu Mini II (Shimadzu, Kyoto,Japan) gas chromatograph (GC) equipped with a methanizer, Po-rapak Q column with 80/100 mesh (Alltech, Deerfield IL, USA)maintained at 40◦C, and flame ionization detector maintained at100◦C. Concentrations were determined relative to commercialstandards of 350, 2040, and 9940 parts per million by volume(ppmv) CO2. Fluxes were calculated as the volume-correctedlinear increase in CO2 concentrations over time per g drypeat.

Extractable noncellular organic and microbial C, N, and P andinorganic N and P were determined using a chloroform (CHCl3)fumigation-extraction technique modified from Voroney et al.(1993), by taking ca. 40 g of peat (wet weight) from each bulksample and splitting it into two 20 g samples. One sample wasplaced in a vacuum desiccator and fumigated with ethanol-freeCHCl3 in the absence of light for 24 h. Chloroform vapor andresidue were then removed through repeated evacuation. Allsamples were sealed in containers containing 0.5 M potassiumsulfate (K2SO4) solution, shaken for 1 h at 200 rpm on an oscil-lating shaker, and filtered through 0.45 µm glass fiber filters. Asub-sample of each extract was oxidized in alkaline persulfateaccording to Williams et al. (1995) for determination of total Nand P in solution as NO3 and ortho-phosphate (PO4). Dissolved

inorganic N (DIN) and P (DIP) were measured as NH4 andnitrite (NO2) + NO3or PO4 using Quik-Chem methods12-107-06-2-A, 10-107-06-2-C, and 10-115-01-1-A on a Latchat FIA+

8000 series continuous flow auto-analyzer (Latchat Instruments,Milwaukee, WI, USA).

Dissolved organic C was measured with a Shimadzu 5050TOC analyzer after pH adjustment to 2.5 and sparging withCO2-free dinitrogen gas (N2) for 10 min. Extractable DOC andDIN were measured directly in K2SO4 extracts from the non-fumigated samples. Extractable organic N and P were calculatedas total extractable N or P from the non-fumigated sub-samplesminus the DIN or DIP. Microbial biomass C, N, and P were cal-culated by subtracting the non-fumigated extractable DOC ortotal N or P from the fumigation-extraction DOC or total N or Prespectively. Because there have been reports of large variationsin C and N extraction efficiency coefficients (KEC, KEN, e.g.,Brookes et al. 1985; Sparling et al. 1990) for different soils, noextraction efficiency coefficients were used in our calculations.All values are expressed per g of dry peat. Relative increases inpools of N and P characterized were calculated as the differencebetween average amounts in treatments minus controls as a per-centage of total added N or P and described as “recovered.” Drybulk density values of 36, 41, 36, and 42 mg cm−3 for the 0–5, 5–10, 10–20, and 20–30 cm depth segments (J. Turunen personalcommunication) were used for volumetric conversions. Respi-ration quotients (qCO2) were calculated as the rate of CO2-Cproduction per microbial biomass C per day (Sakamoto and Oba1994).

Elemental analysis was conducted similar to the methodsused by Basiliko and Yavitt (2001) on a Jarrell Ash inductivelycoupled plasma emission spectrophotometer (ICP-ES ThermoElectron Corp., Waltham, MA, USA). Peat was incubated for24 h in acid-rinsed glass vials in 1:4 peat to deionized water byweight. Approximately 5 ml of water from each container wasfiltered through acid rinsed 0.45 µm filters and frozen at −20◦Cuntil analysis. Concentrations of elements were calculated perg dry peat, and percent recovery of P and K was calculated asabove.

Microbial PLFAs were characterized using a method mod-ified from White and Ringelberg (1998), with an acceler-ated solvent extraction methodology following Macnaughtonet al. (1997). Standards contained fatty acid methyl esters(FAMEs) ranging from C8–C22ω6. Individual fatty acids havebeen used as signatures for various groups of microorganismsacross fertility gradients or in fertilization experiments (e.g.Bardgett et al. 1999; Yao et al. 2000; Grayston et al. 2001). ThePLFAs C18:2ω6c and C18:2ω6t were chosen to represent fun-gal biomass, and PLFAs C11:0, C12:0, C13:0, 2OH C12:0and 3OH C12:0, C14:0, 2OH C14:0 and 3OH C14:0, 14-meth-C15:0, C15:0, 15-meth-C16:0, cyC17:0, C17:0, C18:0,C18:1ω9c, cyC19:0, C20:0 were chosen to represent bacterialbiomass. The quotient of bacterial:fungal PLFAs was used asan indicator of changes in the relative abundance of these twomicrobial groups.

534 N. BASILIKO ET AL.

Carbon Dioxide Flux and Vegetation MeasurementsIn situ CO2 fluxes were measured about 70 times under light

and dark conditions over each of the year 2000 and 2001 growingseasons (May through August), following the method of Bubieret al. (2003). Briefly, measurements were made over 0.36 m2 alu-minum collars in the center of each plot using climate-controlledchambers and a LI-COR 6200 portable photosynthesis system(LiCor Inc., NE, USA). Fluxes were usually calculated over thefirst 2.5 min after placing the chamber on a collar to ensure anair-tight seal. Change in headspace CO2 concentration was mea-sured every 5 s and averaged every 30 s for 2.5 min. The averagesof all flux values measured when photosynthetically active radi-ation (PAR) was greater than 1000 µmol photon m−2 s−1 werechosen to represent maximum net ecosystem exchange of CO2

(NEE). Net ecosystem exchange is the balance between plantphotosynthetic uptake of CO2 and ecosystem respiration (ER).Ecosystem respiration was measured as CO2 efflux under darkconditions created by placing a shroud over the chamber. Max-imum photosynthetic CO2 uptake was the difference betweenNEE and ER. Our values of NEE and photosynthetic uptake aretherefore only representative of activities under reasonably highlight conditions.

Vascular plant species composition was measured by count-ing stem height and number of all species in each collar. Biomassestimates were made with allometric equations derived fromplots harvested for plant biomass outside the fertilization area.Percent cover of mosses was estimated visually and growth mea-sured by a cranked wire method (Clymo 1970).

Statistical AnalysisStatistical analyses using Systat 10 (SPSS Inc. Chicago, IL,

USA) were conducted on results from all determinations ex-cept those from the in situ CO2 fluxes. Effects of fertilizationand depth on nutrients, microbial biomass, CO2 production po-tential, pH, and qCO2 were tested using ANOVA and in cer-tain cases with Tukey post-hoc tests. Analysis of variance andcalculation of 95% confidence intervals were performed usingMinitab (Minitab, State College, PA, USA) to examine differ-ences in NEE, photosynthetic CO2 uptake, and ER among treat-ments. In datasets where distribution of resulting residuals wasnot normal (skewness/SE skewness or kurtosis/SE kurtosis >2or <−2), data were log-transformed and reanalyzed. Howeverfor ease of interpretation, means and standard deviations of non-transformed data are presented. Statistically significant differ-ences were assumed at P ≤ 0.05.

RESULTS

Field ObservationsAfter the first growing season mosses in the 20N+PK plots

were visibly greener than 10N+PK, PK or 0NPK plots. Afterthe second growing season, P. strictum mosses were more abun-dant, growing taller and with wider star-shaped capitula in the

20N+PK plots than in the PK or 0NPK plots. It appeared thatthe taller, denser P. strictum were shading Sphagnum mosses,which were brown colored relative to control and PK plots. To-tal vascular plant biomass was not significantly different amongtreatments, although we did not measure changes in leaf numberor size.

Field Fertilization in the Year 2000After the year 2000 fertilization, CO2 production potential

was significantly lowered with treatment in the 0–5 cm depthsegments, with average 20N+PK rates less than one half of0NPK rates, though significant differences were not observedat any other depths (Figure 1A). At the same depth, micro-bial biomass C increased significantly with treatment, with 80%more biomass C in the 20N+PK treatment (Figure 1B), and bothCO2 production potential and microbial biomass C decreasedsignificantly with depth across all treatments. The concomitanteffects of decreasing CO2 production potential and increasingmicrobial biomass C resulted in a significant qCO2 decreasewith treatment in the 0–5 cm depth segment (Figure 1C).

Microbial biomass N decreased with depth and was signifi-cantly elevated with 10N+PK and 20N+PK treatments in thetop three depth segments and noncellular organic N was signif-icantly elevated in the 0–5 cm depth segments relative to the0NPK control (Figure 1D, Table 1), resulting in significant de-creases in microbial biomass and organic C:N quotients in thetop 10 cm of the peat profile of up to 20 (Table 1). Ammoniumconcentrations were significantly greater in 10 and 20N+PKtreatments than 0NPK control at the 0–5 cm depth with max-imum differences of up to only 5.1 µg N g−1 dry peat, whileNO3 concentrations were significantly lower than controls at5–10 and 10–20 cm depths (Table 1). There was a significantincrease in pH of up to 0.4 units in only the 10–20 cm depthsegment between N+PK additions and 0NPK controls (Table1). Concentrations of P in H2O extracts measured with ICP-ESranged from 0.1 to 1.8 µg P g−1 dry peat and were significantlygreater in both 10 and 20N+PK treatments than in controls withconcentrations up to 158 µg P g−1 dry peat (Table 1). At the20–30 cm depth segment, the 20N+PK treatment had signifi-cantly lower P concentrations than 10N+PK (Table 1). AverageK measured in H2O extracts was greater in 10 and 20N+PKthan 0NPK controls, though with relatively large amounts of Kin controls, differences were not significant (Table 1).

Average PLFA concentrations chosen to represent total mi-crobial, bacteria, and fungal biomass were larger in the 20N+PKtreatment than then 0NPK control, however only the increase infungal biomass was significant (Table 2). This resulted in a de-crease in relative bacterial:fungal quotient.

Calculated total N recoveries in the 10 and 20N+PK treat-ments were 17 and 20%. Most N was detected in microbial andthen organic N in the 0–5 cm depth segments, and calculatedrecovery decreased with depth (Figure 2A and B). Calculated re-covery of NH4 was only based on a significant treatment-control

NUTRIENT EFFECTS IN AN OMBROTROPHIC BOG 535

FIG. 1. Microbial CO2 production potential (A), microbial biomass C (B), microbial metabolic quotient (qCO2) (C), and microbial biomass N (D) in depthsegments of peat (beneath vegetation) following the year 2000 field fertilization. Error bars are the standard deviation of 5 replicates. Significant effects (P < 0.05)of any treatment(s) at individual depths are indicated by an asterisk. Significant variations with depth across all samples are indicated with (+).

difference in the 0–5 cm depth segments and represented lessthan 1% of total added N. In the 10 and 20N+PK plots, 69 and49 percent of added P were recovered in H2O extracts, and dif-ferences between treatments and controls used in calculationswere significant at all depths, while 54 and 53% of added K wasrecovered, though differences were not significant at any depth.There were no consistent patterns of calculated recovery anddepth for P or K (Table 3).

Over the year 2000 growing season, maximum NEE rangedfrom 79 to 136 µg CO2 m−2 s−1, while maximum photosyntheticuptake ranged from 229 to 308, and ER ranged from −139 to−172 µg CO2 m−2 s−1 (Table 4). Although there were no signif-icant differences between treatments and controls, largest val-ues of NEE, PSN and ER were all observed in the 20N+PKtreatments.

Field Fertilization in the Year 2001Following the year 2001 fertilization, CO2 production poten-

tial was significantly elevated in the 0–5 cm depth segment with20N+PK treatment relative to the 0NPK and PK controls (Fig-

ure 3A). There were no significant effects of PK or 20N+PK onmicrobial biomass relative to controls, though average biomasswas slightly larger in the 0–5 cm depth segment (Figure 3B).Both respiration potential and microbial biomass decreased sig-nificantly with depth among all treatments. There were no sig-nificant differences between total or coarse root biomass amongtreatments (data not shown) though fine root biomass was ap-proximately two times larger in the 20N+PK plots than the0NPK or PK controls at the 5–10 and 10–20 cm depths, anddifferences were significant (Figure 3C).

Ammonium concentrations were more than 2 times larger inthe top 2 depth segments of the 20N+PK than PK or 0NPKtreatments with concentrations of up to nearly 200 µg N g−1

dry peat, and NH4 concentrations were significantly smaller inthe two lowest depths of the PK treatments relative to 0NPK(Figure 3D). Similarly, NO3 concentrations were significantlysmaller in the PK than 0NPK controls, though no significantdifferences were measured between 20N+PK treatments and0NPK controls, and concentrations never exceeded 2 µg N g−1

dry peat (Table 5). In the 20N+PK treatment, microbial N was

536 N. BASILIKO ET AL.

TABLE 1K2SO4 extractable NO3-N, NH4-N, extractable organic N, extractable organic C:N quotient, microbial biomass C:N quotient, pH,

and H2O-soluble P and K in depth segments of peat following the year 2000 field fertilization

Treatment Treatment

0NPK 10N+PK 20N+PK 0NPK 10N+PK 20N+PK

NO3-N (µg g−1) +Depth+ NH4-N (µg g1)1.5 (0.3) 1.6 (0.5) 1.2 (0.3) 0–5∗ 2.9 (0.6) 8.3 (3.4) 6.8 (2.6)3.0 (0.5) 1.8 (1.0) 1.8 (0.3) ∗5–10 3.2 (0.4) 12.9 (9.5) 5.2 (0.8)2.6 (0.1) 1.4 (0.2) 1.0 (0.1) ∗10–20 4.6 (2.8) 3.6 (1.0) 2.7 (0.3)1.1 (0.6) 1.7 (0.5) 1.1 (0.6) 20–30 3.8 (0.5) 4.0 (0.9) 3.6 (0.4)

Extractable organic N (µg g−1) Depth Extractable organic C:N quotient45 (6.7) 96 (12) 107 (33) ∗0–5∗ 40 (2.0) 23 (1.1) 20 (1.7)57 (4.3) 59 (30) 83 (11) 5–10∗ 33 (3.2) 20 (4.1) 26 (2.0)55 (10) 44 (5.5) 40 (4.0) 10–20 27 (2.8) 22 (4.9) 27 (2.5)55 (1.4) 40 (4.9) 52 (10) 20–30 23 (1.5) 20 (3.8) 24 (1.5)

Microbial C:N quotient Depth+ pH40 (2.0) 23 (1.1) 20 (1.7) ∗0–5 4.4 (0.2) 4.7 (0.2) 4.6 (0.1)33 (3.2) 20 (4.1) 26 (2.0) ∗5–10 4.3 (0.1) 4.5 (0.3) 4.4 (0.1)27 (2.8) 22 (4.9) 27 (2.5) 10–20∗ 4.0 (0.1) 4.4 (0.1) 4.2 (0.1)23 (1.5) 20 (3.8) 24 (1.5) 20–30 4.0 (0.1) 4.2 (0.1) 4.0 (0.1)

P (µg g−1) Depth K (µg g−1)1.8 (0.7) 139 (29) 158 (35) ∗0–5 421 (178) 624 (326) 475 (165)0.4 (0.5) 158 (45) 133 (11) ∗5–10 349 (129) 571 (166) 447 (132)0.3 (0.5) 95 (10) 87 (12) ∗10–20 379 (127) 444 (205) 441 (102)0.1 (0.2) 109 (27) 27 (30) ∗20–30 286 (185) 420 (233) 532 (223)

Mass is based on dry peat. Values in parentheses are the standard deviation of 5 replicates. Significant effects (P < 0.05) of any treatment(s)at individual depths are indicated with ∗and those with depth across all samples with +.

elevated up to 2 times the concentrations measured in 0NPKor PK treatments (Figure 3E), and microbial biomass C:N wassignificantly lower in all depths except 10–20 cm, though dif-ferences were much smaller than measured after the first year(Tables 5, 1).

Organic N concentrations were significantly smaller in thetwo lowest depth segments of the PK treatments relative to the

TABLE 2Microbial phospholipid fatty acids (PLFAs) in the 0–5 cm

depth segment of peat following the year 2000 fieldfertilization, based on dry peat mass

Treatment

PLFA (ng g−1) 0NPK 20N+PK

Total microbial 333 (40) 440 (181)Total bacterial 230 (26) 290 (141)Total fungal∗ 103 (14) 150 (32)Bacterial: fungal 2.2 (0.1) 1.8 (0.4)

Values in parentheses represent the standard deviation of 5 replicates.Significant differences (P ≥ 0.05) are indicated with an asterisk.

0NPK controls, but there were no significant differences in eitherorganic N or C:N between 20N+PK treatment and 0NPK con-trols (Table 5). Phosphate concentrations were significantly, andup to 10 times, greater in PK and 20N+PK relative to controlsamong all depths (Table 5). Microbial biomass P and organic P

TABLE 3Calculated recovery of added P and K in H2O extracts after the

year 2000 field fertilization

Treatment Treatment

10N+PK 20N+PK 10N+PK 20N+PK

Depth (cm) P (%) K (%)

0–5 13 14 12 35–10 16 14 15 710–20 17 16 8 720–30 23 6 19 35Total 69 49 54 52

Values were calculated as the relative increase in either mean P orK between the controls and treatment as a percentage of total P or Kadded.

NUTRIENT EFFECTS IN AN OMBROTROPHIC BOG 537

FIG. 2. Calculated recovery of N or P in microbial biomass, extractable organic, and inorganic forms following fertilization calculated as differences betweenmeans of treatments and controls (five replicates) expressed as a percentage of added N or P for years 2000 or 2001. Asterisks indicate mean differences that weresignificantly different from controls. Total recovery values also include negative values in cases where average mass calculations of N or P species in treatmentswere less than in controls.

were elevated in PK and 20N+PK treatments (Table 5, Figure3F), and although differences were not significant, average mi-crobial biomass P was greater in 20N+PK than PK treatmentsacross all depths (Figure 3F).

Extractable organic N:P quotients decreased by up to 9 timeswith PK or 20N+PK fertilization relative to controls, and micro-bial biomass N:P quotients decreased throughout all depths, butby only up to approximately 3 times (Table 5). Extractable DOCdecreased slightly with PK additions relative to 0NKP in lowerdepth segments, though there were no significant differences inthe 0–5 cm depth segment (Table 5). Average pH increased with20N+PK at certain depths, though increases were never morethan 0.5 units (Table 5).

Total calculated N recovery after 2 years of fertilization with20N+PK treatment was 16% of the added amount, with most Nmeasured in the surface 0–5 cm depth segment (Figure 2C). Lessthan 1% of the amount of N was inorganic, present as NH4 in the

5–10 cm depth segment, whereas the largest calculated recoveryof N was in the microbial form followed by the noncellularorganic form. Calculated recoveries of amounts of N added werebased on statistically significant differences between treatmentsand controls in all but the 20–30 cm depth segment. More than100% of the amount of added P was recovered in PO4, microbial,and noncellular organic forms from the 20N+PK plots, with atleast 22% of the amount of P added recovered from each of thefour depth segments (Figure 2D), and in contrast to with N, therewas no clear decreasing depth-recovery pattern. Most additionalP relative to controls was present as PO4, followed by microbialand organic pools. Patterns were similar for P recovery in PKplots, though total recovery of the amount of added was 89%,with less P recovered at the top three depths (25, 19, and 18%)and the largest recovery at the lowest depth (27%).

We observed no significant differences between the measure-ments of CO2 production potential, microbial biomass C, and

538 N. BASILIKO ET AL.

TABLE 4In situ seasonal mean CO2 fluxes (µg CO2 m−2 s−1), including net ecosystem CO2 exchange (NEE) and photosynthetic CO2

uptake (photosynthesis) at measured at high light levels (PAR > 1000 µ mol photon m−2 s−1) and ecosystem respiration (ER)among plots in year 2000 and 2001

2000 2001

CO2 flux 0NPK 10N+PK 20N+PK 0NPK PK 20N+PK

NEE 97 (110) 79 (106) 136 (123) 44 (101) 114 (123) 75 (114)

DF 5; F-ratio 1.76; P NS DF 6; F-ratio 2.29; P NSPhotosynthesis 238 (158) 229 (163) 308 (167) 264 (92) 392 (92) 374 (233)

DF 5; F-ratio 1.98; P NS DF 6; F-ratio 10.21; P < 0.05ER −136 (79) −145 (75) −172 (88) −207 (92) −260 (84) −286 (114)

DF 5; F-ratio 1.39; P NS DF 6; F-ratio 16.19; P < 0.05

Positive values represent net CO2 uptake and negative values represent net loss. Standard deviations are in parentheses and degrees of freedom(DF) F-statistic ratios (F-ratio) and P values from one-way ANOVA are grey (NS = not significant).

TABLE 5K2SO4 extractable NO3-N, extractable organic N, extractable organic C:N, microbial biomass C:N, PO4-P, extractable organic P,

extractable organic N:P, microbial biomass N:P, pH, and DOC in peat following the year 2001 field fertilization

Treatment Treatment

0NPK PK 20N+PK 0NPK PK 20N+PK

NO3-N (µg g−1) Depth+ Extractable organic N (µg g−1)1.8 (0.1) 1.4 (0.2) 1.8 (0.5) 0–5 192 (67) 143 (40) 169 (62)1.5 (0.2) 1.3 (0.3) 1.6 (0.3) 5–10 76 (15) 87 (59) 89 (15)1.8 (0.2) 1.2 (0.2) 1.6 (0.1) ∗10–20∗ 110 (54) 47 (11) 85 (6)1.9 (0.5) 1.3 (0.2) 1.9 (0.2) ∗20–30∗ 83 (16) 56 (15) 75 (3)

Extractable organic C:N quotient Depth Microbial C:N quotient16 (4.3) 14 (1.2) 15 (4.5) 0–5∗ 12 (2.7) 11 (2.9) 6.7 (0.5)19 (1.2) 16 (2.9) 17 (2.6) 5–10∗ 10 (1.9) 12 (1.8) 7.1 (0.8)16 (4.7) 17 (0.9) 14 (0.4) 10–20 11 (5.8) 12 (1.2) 6.8 (0.6)16 (1.8) 18 (4.7) 13 (0.9) 20–30∗ 8.4 (0.8) 10 (1.7) 6.7 (0.8)

PO4–P (µg g−1) Depth+ Extractable organic P (µg g−1)21 (12) 288 (67) 334 (98) ∗0–5∗ 11 (1.2) 64 (34) 61 (22)

6.8 (5.3) 202 (67) 229 (88) ∗5–10∗ 6.0 (1.6) 31 (20) 26 (8)7.8 (7.2) 187 (20) 186 (27) ∗10–20 7.7 (4.5) 34 (27) 23 (4)4.8 (1.1) 218 (48) 183 (23) ∗20–30∗ 3.4 (0.4) 17 (7) 22 (6)

Extractable organic N:P quotient Depth Microbial N:P quotient18 (5.5) 2.5 (0.7) 2.8 (0.3) ∗0–5∗ 2.6 (0.2) 2.0 (0.4) 2.3 (0.3)13 (2.9) 2.8 (0.6) 3.5 (0.8) ∗5–10∗ 2.7 (0.1) 1.5 (0.3) 2.0 (0.4)17 (6.6) 1.9 (0.8) 3.8 (0.8) ∗10–20∗ 2.7 (0.9) 1.2 (0.3) 1.4 (0.1)25 (6.6) 4.1 (2.7) 3.5 (0.9) ∗20–30∗ 3.9 (0.9) 1.1 (0.2) 1.4 (0.4)

PH Depth+ Extractable DOC (µg g−1)3.8 (0.1) 4.0 (0.1) 4.3 (0.1) ∗0–5 2.8 (0.5) 2.0 (0.4) 2.3 (0.4)3.9 (0.1) 4.0 (0.1) 4.2 (0.1) ∗5–10 1.4 (0.2) 1.3 (0.5) 1.5 (0.4)3.9 (0.2) 4.0 (0.2) 4.2 (0.2) ∗10–20∗ 1.6 (0.4) 0.8 (0.2) 1.2 (0.1)3.9 (0.2) 4.0 (0.1) 4.2 (0.1) 20–30∗ 1.4 (0.1) 1.0 (0.2) 1.0 (0.1)

Mass is based on dry peat. Values in parentheses represent the standard deviation of 5 replicates. Significant effects (P < 0.05) of anytreatment(s) at individual depths are indicated by∗. Significant variations with depth across all samples are indicated with (+).

NUTRIENT EFFECTS IN AN OMBROTROPHIC BOG 539

FIG. 3. Microbial CO2 production potential (A), microbial biomass C (B), fine root biomass (C), K2SO4 extractable NH4-N (D), microbial biomass N (E) andmicrobial biomass P (F) in depth segments of peat following the year 2001 field fertilization. Error bars are the standard deviation of 5 replicates. Significant effects(P < 0.05) of any treatment(s) at individual depths are indicated by an asterisk. Significant variations with depth across all samples are indicated with (+).

nutrients in samples with or without roots with most treatmentsat most depths. In individual samples, CO2 production potentialwas often smaller with roots removed, though among all treat-ments and depths, bulk and root-free samples had potentials thatwere correlated (root-free CO2 production = 0.65 × bulk peat

CO2 production +0.07, r2 = 0.50). Among replicates, differ-ences were only significant in the 10–20 cm depth segment in the0NPK and PK treatments, and the 20–30 cm depth segment in thePK treatment. There were no differences in microbial biomassC or NH4 between bulk and root-free samples at any depth or

540 N. BASILIKO ET AL.

treatment, though in most instances (0NPK 0–5 and 5–10 cm;PK 10–20 and 20–30 cm; 10N+PK 5–10, 10–20, and 20–30 cm),NO3 concentrations were greater in root-free samples.

Over the year 2001 growing season there were no significantdifferences in NEE between treatments and controls, rangingfrom 44 to 114 µg CO2 m−2 s−1 (Table 4). However maximumphotosynthetic CO2 uptake and ER showed a different pattern.Photosynthetic uptake in the PK and 20NPK plots was signifi-cantly greater (374 – 392 µg CO2 m−2 s−1) than controls (264 µgCO2 m−2 s−1). Similarly, ER was greater (more negative) in thePK and 20NPK plots (−260 to −286 µg CO2 m−2 s−1) relativeto controls (−207 µmol CO2 m−2 s−1). Because photosynthesisand respiration offset each other in the NEE measurement, thismay explain the lack of difference in NEE among treatments. Itshould also be noted that ER was larger in year 2001 comparedto year 2000 in the controls owing to a lower water table (Bubieret al. 2003).

DISCUSSION

Field Fertilization in the Year 2000Aerial nutrient additions (i.e., applied from above) in the field

generally caused effects at the surface of the peat profile. De-creased CO2 production potential and concomitant increasedmicrobial biomass formation occurred in upper layer of the peatprofile with increasing N (with P and K) addition after one sea-son of fertilization (Figure 1A). This is similar to Williams andSilcock’s (1997) observation in a UK bog, where a substrate-induced respiration method was utilized to characterize micro-bial biomass C. Of the mechanisms suggested by Agren et al.(2001) potentially linking inorganic N additions to decreasedorganic matter decomposition, we suggest a change in the mi-crobial community as the most likely. We observed decreasedqCO2 in the upper depths of the peat profile and a compositionalchange in community structure with increased fungal biomass.Because microbial biomass C increased with increasing N+PKaddition, decreased microbial growth does not appear to have oc-curred (Figure 1B). The formation of recalcitrant organic matter,primarily amino-quinones (Agren et al. 2001), as a result of NH4

additions may have occurred, as we observed increasing DONwith N addition in the upper depth segment of the peat pro-file (Table 1). However it seems unlikely that decreased organicmatter bioavailability would lead to larger microbial biomass.

We suspect that decreased decomposition and increased mi-crobial biomass C occurred because of N additions and not Por K, because we observed consistently increasing microbialbiomass and decreasing CO2 production potential between the0NPK, 10N+PK, and 20N+PK treatments, though having notsampled from the PK plot in the year 2000 for comparison, wecannot rule out the potential combined effects of N, P, and K.Because P and K addition levels were identical in the 10 and20N+PK treatments, differences can be related to N addition;however it is important to note that these are effects of N whenhigh concentrations of P and K were deposited. We only mea-

sured PLFAs in the 0NPK and 20N+PK but not 10N+PK plotsand therefore cannot speculate on which nutrients were respon-sible for the compositional changes in the microbial communitystructure. Increased fungal biomass and decreased CO2 produc-tion are consistent with the observations of Holland and Coleman(1987), who reviewed literature on assimilation efficiencies andconcluded that soil fungi assimilate more C and respire less Cas CO2 than bacteria, and with Sakamoto and Oba (1994) whoreported a high negative correlation between qCO2 and fungal:bacterial biomass ratios.

Significant increases in N occurred primarily in the noncel-lular organic and microbial forms, and the increases observedin NH4 in the upper segment of the profile represented less than1% of added N. We suspect that the Sphagnum mosses quicklysequestered added N and N was transferred to the noncellularorganic and microbial forms through exudation of DON, or mi-crobial uptake of these exudates or microbial assimilation fromdecomposing newly formed moss tissues. Fenner et al. (2004)recently demonstrated that Sphagnum mosses transfer a largeportion of recent photosynthates to the dissolved form in a mat-ter of hours that may be available for uptake by microorganisms.

Uptake of N by microorganisms resulted in significant de-creases in biomass C:N quotients in the surface of the peat pro-file, ranging from 40 in the controls to 20 in treatment plots.Microbial biomass C:N quotients calculated from bacterial andfungal cultures are much smaller than those we encountered,ranging from 3 to 15 (Paul and Clark 1996). Johnson et al. (2003)suggested that calculation techniques and a lack of previous un-derstanding of soil microbial communities in northern ecosys-tems explained large C:N quotients, rather than inaccuracies inthe fumigation-extraction method resulting from variability inmoisture content or organic matter properties of the soils. Starkand Hart (2003) criticized high biomass C:N quotients in a sub-arctic heath ecosystem. For statistical analyses, we calculatedmicrobial C:N quotients for individual samples and presentedmeans, whereas those presented by Stark and Hart (2003) werecalculated on the basis of mean biomass C and N. Recalculationwith this method decreased our largest C:N quotient to 30, nearlywithin the range (20–29) that Stark and Hart (2003) also considerto be large but realistic for certain soil microbial communities.

In the absence of N limitation, P has been suggested to bethe next limiting nutrient to primary production in peatlands(Aerts et al. 1992). Our calculated total recovery of P in H2Oextracts was large in 10N+PK and 20N+PK treatments afterone season of fertilization and its recovery did not decrease withdepth, as with N, suggesting that the vegetation at Mer Bleuewas not P limited, at least under simultaneous N availability. If Pwere limiting, we would have expected greater retention closerto the living vegetation and less P to be present in water-solubleand potentially mobile form. Water extractable K was present inlarge amounts in controls relative to other ombrotrophic peat-lands, and we suspect it was not limiting primary production.We detected a mean of 8.0 mg K L−1 in H2O extracts from alldepths of control plots compared to reports of <2.0 by Basiliko

NUTRIENT EFFECTS IN AN OMBROTROPHIC BOG 541

and Yavitt (2001) and <1.0 mg K L−1 by Robert et al. (1999) inpore water from 7 north American peatlands. We did not measuretotal N content of vegetation or peat, though based on ambientdeposition rates in context of Vitt et al. (2003), previous work onassimilation and retention of N in vegetation and peat in unpol-luted regions by Li and Vitt (1997), and a strong depth-recoverydependence of soluble forms of N, but not P, we suspect thatmuch of the added N was present in plant and peat tissues.

There was little evidence of increased Sphagnum growth inthe 10N+PK or 20N+PK treatments after the year 2000 fertil-ization (cranked wire method, Tim Moore, personal communi-cation), however the 20N+PK moss was visibly greener thancontrols or 10N+PK plots, indicating that plants were affectedby nutrient additions.

Despite reduced CO2 production potential in the surface peatprofile there were no significant differences in average ER orNEE measured over the entire growing season. Effects seen inthe laboratory incubations represent conditions at a single pointin time after an entire season of fertilization, and were observedin only a very small portion of the peat profile sampled from oneplot. Average in situ fluxes however represent both plant activityand decomposition of the entire peat profile over a longer periodof time, likely explaining why reduced rates of decompositionwith fertilization did not translate into slower ER or NEE.

Field Fertilization in the Year 2001In contrast to the year 2000, decomposition measured as CO2

production potential was elevated in the upper depth segment of20N+PK treatments relative to both the 0NPK controls and thePK following the second year of fertilization, while there were nosignificant differences in microbial biomass C. Fine root biomassincreased significantly in the 20N+PK plots, which could havesupplied a labile source of organic matter contributing to en-hanced decomposition. Increases were observed, however, inlower three depth segments, but not the 0–5 cm segment. Poly-trichum strictum was clearly shading out Sphagnum mosses inthe 20N+PK plots, potentially causing enhanced decompositionof the Sphagnum or decomposition of newly formed P. strictumbiomass, which may be more readily decomposable than Sphag-num tissues. Berendse et al. (2001) reported that inorganic Nfertilization caused a similar shift in moss species in Europeanpeatlands and predicted that this would decrease C sequestrationdue to increased tissue decomposability.

Enhanced CO2 production could also have arisen from in-creased organic matter exudates from the moss layer, althoughwe did not measure increased DOC in the upper depth seg-ment of the profile. In contrast to our findings, Tommasen et al.(2004) did not observe increased peat decomposition followingthree years of N fertilization in an Irish bog, owing to decreasedpH presumably occurring as a result of increased H+ mobiliza-tion from cation exchange sites at high NH4 concentrations. Al-though we did observe significant increases in NH4 after the year2001 fertilization, pH increased significantly, although slightly,with N addition.

While it is clear that increased N deposition can alter peat de-composition, and potentially affect ER, Aerts et al. (2001) foundthat effects of N fertilization are subject to differences in envi-ronmental variability. Given environmental differences betweenthe years 2000 and 2001 at Mer Bleue, our finding of decreasedand then increased decomposition occurring with N fertiliza-tion must be interpreted as having occurred under relatively wetand then relatively dry conditions. Tommasen et al. (2004) con-cluded that N deposition was more important than drying tothe growth of non-peat forming plants in peatlands, but did notexplicitly investigate Polytrichum mosses. Polytrichum strictumcan tolerate and grow under much drier conditions than Sphag-num, and we cannot eliminate the possibility that enhanced P.strictum growth was not due to a combination of high N loadingand relatively dry conditions.

Significantly less NH4, NO3, and organic N in each of the seg-ments at the lower 2 depths after PK treatment than after 0NPKmay indicate that added P was increasing plant uptake and im-mobilization from N in the soluble pools, although differenceswere small. The majority of the amount of additional recov-ered N relative to controls was in the microbial biomass, thoughin contrast to the year 2000, NH4 and not organic N consti-tuted the remainder of the calculated N recovery. This may haveresulted from enhanced microbial activity mineralizing a largerportion of organic N that was not held by the vegetation. Slightlymore than 100% of the amount of added P relative to controlswas detected primarily in inorganic and then in microbial forms.Though additional P was present in the microbial biomass in PKand 20N+PK treatments and biomass N:P quotients decreasedat all depths, this microbial physiological change with P andK addition did not affect microbial activity measured as CO2

production potential. Williams and Silcock (2001) investigatedthe effects of N fertilization on P and reported no significanteffects of fertilization on microbial biomass P in a UK peat-land. We observed greater average microbial P in 20N+PK thanPK treatments throughout depth, but in no instances were thesedifferences significant, in agreement with William and Silcock(2001). Based on nutrient concentrations after 2 seasons of fer-tilization, the vegetation in the 20N+PK treatment still appearedto be N limited, because calculated N recovery was relativelysmall and exhibited depth dependence, while P recovery waslarge and did not exhibit depth dependence.

In contrast to nutrient recovery patterns, in situ CO2 fluxesin the year 2001 suggest that P or K were limiting nutrients,with elevated photosynthetic uptake and ER in both PK and20N+PK treatments relative to controls (Table 4). At least forthe 20N+PK treatments, the ER data are supported by increasedmicrobial CO2 production measured in the lab after the year 2001field season (Figure 3).

CONCLUSIONSIn relatively unpolluted sites such as Mer Bleue, the tight

coupling of activities of plants and microorganisms influencesthe effects of increased N additions, and the vegetation mediates

542 N. BASILIKO ET AL.

effects on the microbial community and litter and peat decompo-sition. Nitrogen is cycled primarily in the organic forms, in con-trast to P, of which a large portion can exist in the inorganic form.Measurements dealing with N and P in the lab and field experi-ments yielded contradictory results regarding nutrient limitation.We cannot rule out that these findings reflect discrepancies inexperimental design, especially because the laboratory resultswere derived from very limited spatial and temporal sampling.We characterized potential controls on nutrient and C dynamics,however given the biogeochemical complexity of peatlands andin light of discrepancies revealed between some of our measure-ments, we may have not yet identified all of the crucial systemparameters driving these transformations. Even in light of inher-ent system complexity, we conclude that nutrient deposition canlead to decreased rates of microbial CO2 production, though atlarge deposition levels these effects are short-lived, likely due toa concomitant shift in moss-species.

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