PhD thesis_Shimon_final_combined

F A C U L T Y O F S C I E N C E
U N IV E R S I T Y O F C O P E N H A G E N
PhD thesis
Shimon Ozeri Ginzburg
Nitrogen deposition effects on soil carbon dynamics in temperate forests
Academic advisors:
Per Gundersen, Professor
Lars Vesterdal, Associate Professor
Submitted: 15 July 2014

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Title: Nitrogen deposition effects on soil carbon dynamics in temperate forests
Author: Shimon Ozeri Ginzburg
Academic advisors: Per Gundersen (main supervisor), Lars Vesterdal (co-supervisor)
Citation: Ginzburg S. (2014) Nitrogen deposition effects on soil carbon dynamics in temperate
forests. PhD thesis. Department of Geosciences and Natural Resource Management. University of
Copenhagen. 195 pp.
Submitted: 15 July 2014
Cover photo: The forest edge investigated in Sønder Omme, Denmark
This report is published at http://ign.ku.dk

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PREFACE AND ACKNOWLEDGEMENTS.....................................................................................................................................4
LIST OF PAPERS.................................................................................................................................................................................5
ABSTRACT ...........................................................................................................................................................................................6
DANISH ABSTRACT / DANSK RESUMÉ........................................................................................................................................8
LIST OF ABBREVIATIONS .............................................................................................................................................................10
THE AIM OF THE PROJECT..........................................................................................................................................................11
GENERAL INTRODUCTION...........................................................................................................................................................12
NITROGEN DEPOSITION ......................................................................................................................................................................12
ECOSYSTEM PARTITIONING OF SEQUESTERED C AND IMPACTS OF N DEPOSITION .................................................................................12
NITROGEN DEPOSITION EFFECTS ON ABOVE- AND BELOWGROUND C INPUTS........................................................................................14
EFFECTS ON SOIL C OUTPUTS .............................................................................................................................................................15
EFFECTS ON SOIL C STOCKS ...............................................................................................................................................................15
WHY STUDY N DEPOSITION EFFECTS ON FOREST C SEQUESTRATION?..................................................................................................16
NITROGEN DEPOSITION EFFECTS ON SOM TURNOVER IN A WARMER WORLD .......................................................................................17
FOREST EDGES...................................................................................................................................................................................18
METHODOLOGY..............................................................................................................................................................................19
THE FOREST EDGE SITES.....................................................................................................................................................................19
Experimental design – forest edges.............................................................................................................................................23
THE KLOSTERHEDEN N-ADDITION EXPERIMENT .................................................................................................................................23
SAMPLING AND ANALYSIS..................................................................................................................................................................25
Microclimate at the edge sites.....................................................................................................................................................25
Throughfall .................................................................................................................................................................................26
Foliar litter .................................................................................................................................................................................27
Forest floor and mineral soil sampling.......................................................................................................................................28
Fine roots....................................................................................................................................................................................29
Nitrate leaching ..........................................................................................................................................................................30
SOM turnover .............................................................................................................................................................................31
Temperature sensitivity...............................................................................................................................................................33
RESULTS.............................................................................................................................................................................................34
FOREST EDGE EFFECTS ON N DEPOSITION AND N AVAILABILITY..........................................................................................................34
NITROGEN DEPOSITION EFFECTS ON ABOVEGROUND C INPUTS ............................................................................................................37
NITROGEN DEPOSITION EFFECTS ON BELOWGROUND C INPUT BY ROOTS AND EM ...............................................................................38
NITROGEN DEPOSITION EFFECTS ON SOM TURNOVER.........................................................................................................................40
NITROGEN DEPOSITION EFFECTS ON SOC STOCKS ..............................................................................................................................42
NITROGEN DEPOSITION EFFECTS ON Q10 OF SOIL RESPIRATION ............................................................................................................45
SUMMARY DISCUSSION.................................................................................................................................................................47
NITROGEN DEPOSITION AND N AVAILABILITY.....................................................................................................................................47
HOW DOES ELEVATED N DEPOSITION AFFECT ABOVEGROUND C INPUTS INTO THE SOIL?......................................................................49
HOW DOES ELEVATED N DEPOSITION AFFECT BELOWGROUND C INPUTS INTO THE SOIL? .....................................................................49
HOW DOES ELEVATED N DEPOSITION AFFECT THE DECOMPOSITION (C OUTPUTS) OF SOM? ................................................................50
WHAT IS THE NET EFFECT OF ELEVATED N DEPOSITION ON SOC STOCKS?...........................................................................................51
HOW DOES ELEVATED N DEPOSITION AFFECT THE SHORT-TERM TEMPERATURE SENSITIVITY OF SOM DECOMPOSITION?......................53
CONCLUSIONS AND PERSPECTIVES .........................................................................................................................................55
CARBON SEQUESTRATION ..................................................................................................................................................................55
ENVIRONMENTAL MANAGEMENT .......................................................................................................................................................55
FOREST EDGES AS A PLATFORM FOR STUDYING C AND N DYNAMICS ...................................................................................................56
FUTURE RESEARCH ............................................................................................................................................................................57
BIBILIOGRAPHY..............................................................................................................................................................................59
Paper I……………………………………………………………………………………………………………………………………………………..
Paper II……………………………………………………………………………………………………….…………………………………………...
Paper III…………………………………………………………………………………………………………………………………………………...

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Preface and acknowledgements
This PhD dissertation has been submitted for the attainment of a PhD degree to the Department of
Geosciences and Natural Resource Management, Faculty of Science, University of Copenhagen,
Denmark. This study was funded by the VILLUM Foundation and Nordic Council of Ministers
through the Forest Soil C-sink Nordic Network project and by the Science PhD School. The field
work at Thetford was funded by Forest Research, UK and by the EU COST Action FP0803
(Belowground carbon turnover in European forests) as a short scientific mission (STSM).
This PhD is part of the Forest Soil C-sink Nordic Network project aiming to improve the
understanding of factors affecting the accumulation and loss of forest soil organic carbon.
First of all I would like to thank my supervisors Per and Lars for all their support through the course
of my work. Per, equipped with your calmness and experience I couldn’t go wrong! Lars, through
your maddening attention to details I learned the essence of scientific writing. Both of you inspired
me by your passion and enthusiasm for forest ecology even when I was weary.
I am also very grateful for the assistance in field and lab work given by Preben Frederiksen, Xhevat
Haliti, Mads M. Krag and Allan O. Nielsen, Maria Bartolomé Criado, Lei Liu, Maxwell Owusu-
Twum, Mikkel Eeg, Cæcilie Gervin, Jeremie Chevillotte and Jean Francois Schiratti, your humor
made sampling and analysis fun.
I wish also to acknowledge the support provided by Dr. Elena Vanguelova during my visit to Forest
Research, UK and field work to Thetford and to Dr. Björn Erhagen from SLU Umeå for looking
after my incubated samples.
Finally, I would like to thank my dear wife Rikke for backing me up when the times got rough. It is
much appreciated.

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List of papers
This PhD thesis includes the following three papers that are referred to in the text.
I. Ginzburg S, Vesterdal L, Vanguelova E, Hansen K, Schmidt KI, Gundersen P (in review)
Does nitrogen deposition affect soil carbon stocks? – Evaluation of five nitrogen gradients
in temperate forest edges. Global Change Biology
II. Ginzburg S, Schmidt KI, Kjøller R, Vesterdal L, Gundersen P (in review) Changes in soil
carbon flows and sequestration rates following long-term chronic nitrogen additions to a
Norway spruce forest. Forest Ecology & Management
III. Ginzburg S and Gundersen P (in revision) Elevated N deposition moderates the temperature
feedback on decomposition in forest soils. Nature Communications

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Abstract
Soils contain the largest fraction of terrestrial carbon (C). Understanding the factors regulating the
decomposition and storage of soil organic matter (SOM) is essential for predictions of the C sink
strength of the terrestrial environment in the light of global change. Elevated long-term nitrogen (N)
deposition into forest ecosystems has been increasing globally and was hypothesized to raise soil
organic C (SOC) stocks by increasing forest productivity and by reducing SOM decomposition.
Yet, these effects of N deposition on forest SOC stocks are uncertain and largely based on
observations from high dose-N fertilization experiments. Here, I present a synthesis of results from
three studies aiming at elucidating the effects of long-term N deposition on SOC stocks and fluxes
in temperate conifer forests. In two of the studies, short N deposition gradients occurring naturally
in forest edges were used to study the effects of varying N deposition load on SOC stocks and
fluxes as well as on the temperature sensitivity of SOM respiration. In a third study, the effects of
20 years of continuous experimental N addition (35 kg N ha-1
year-1
) on soil C budget were
investigated.
Our general hypotheses were that elevated N deposition will: i) increase SOC stocks owing to
positive effect of N on litterfall C inputs combined with negative effect on SOM decomposition
regardless of negative effects on belowground C inputs by roots and associated mycorrhiza; ii)
reduce the temperature sensitivity of SOM heterotrophic respiration.
The five forest edges investigated exhibited N deposition gradients decreasing from the edges
toward the interior of the stands. Thus, increasing distance from the edge was used as proxy for
decreasing N deposition. At the two edges receiving intermediate-N deposition rates (≤ 23 kg N ha-1
year-1
), forest floor SOC stocks tended to decrease with distance from the edge while a decade of
experimental N additions insignificantly increased the size of the humus (H) layer providing weak
evidence for some positive effect of N deposition on SOC stocks. The three edges receiving higher
N deposition rates (> 29 kg N ha-1
year-1
) were N-saturated where SOC stocks in the top soil were
either unchanged or negatively affected by elevated N deposition. Similarly, forest floor SOC
stocks were unchanged following two decades of experimental-N additions.

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Aboveground C inputs by needle litterfall were generally not significantly affected by N deposition
at the edge sites but tended to increase with increasing distance from the edge in two of the N-
saturated sites. The experimental N additions resulted in reduced C inputs by foliar litter relative to
control concomitant with reduced tree and moss growth. At three edges, fine-root biomass tended to
increase with distance from the edge while over a decade of N additions reduced the means of fine-
root biomass, ectomycorrihizal (EM) mycelial production and species composition insignificantly
compared with control suggesting reduced belowground C inputs under elevated N deposition.
At two edge sites, forest floor C outputs by respiration tended to decrease with decreased forest
floor C/N and distance from the edge indicating positive effect of elevated N deposition on SOC
sequestration. Correspondingly, N-enriched litter incubated in litterbags had significantly lower
late-stage decomposition rates compared with control litter. However, potential respiration of forest
floor and mineral soil was overall unaffected by the experimental N-additions. A temperature
treatment of forest floor samples taken from one edge site revealed decreasing respiration rates and
temperature sensitivity (Q10) of labile and recalcitrant SOM with increasing forest floor C/N ratio.
I conclude that N deposition at rates typical to agriculturally intensified regions in north-western
Europe may reduce the decomposition rate of SOM thereby positively affecting SOC sequestration
but at the same time above- and belowground C inputs also decrease resulting in no change in SOM
stocks. Moreover, elevated N deposition may reduce the potential positive feedback of SOM
decomposition on global atmospheric CO2 concentrations. These results have implications for
modelling the carbon sink-strength of temperate forests under global change.

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Danish Abstract / Dansk resumé
Jordbunden indeholder den største andel af terrestrisk kulstof (C). Set i lyset af globale
klimaforandringer er det essentielt at forstå de faktorer, der regulerer omsætning og lagring af
jordbundens organiske materiale (SOM) for at kunne forudsige miljøets evne til at akkumulere C.
Globalt set har den langvarige kvælstof (N)-deponering til skov-økosystemer været stigende. Dette
kan forårsage stigninger i jordens organisk C (SOC) pulje ved at øge skovens produktivitet og
reducere omsætningen af SOM. Denne sammenhæng er dog usikker og primært baseret på
observationer fra høj dosis N gødskningseksperimenter. Jeg vil her præsentere en sammenfatning af
resultaterne fra tre undersøgelser, der har haft som formål, at belyse effekten af den langvarige N-
deponering på SOC-indhold og -flukse i tempererede nåleskove. I to af undersøgelserne blev den
naturlige gradient for korttidsdeponering af N i skovkanten brugt som basis for at undersøge
effekten af forskellige N-deponeringsniveauer på SOC-indhold og -flukse samt på
temperaturfølsomheden af SOM-respirationen. I den tredje undersøgelse blev effekten på
jordbundens C-budget af 20 års kontinuer eksperimentel tilførsel af N (35 kg N ha-1
year-1
)
undersøgt.
Vores overordnede hypotese var, at en forhøjet N-deponering vil: i) øge SOC-indholdet som følge
af den positive effekt N har på nåle-litter kombineret med den negative effekt på SOM-
omsætningen, på trods af negative effekter for C-input fra rødder og tilhørende mycorrhiza; ii)
reducere temperaturfølsomheden af den heterotrofe SOM-respiration.
De fem undersøgte skovkanter viste aftagende N-deponeringsgradienter fra skovkanten og ind mod
skovens indre. Derfor blev øget afstand fra skovkanten brugt som en proxy for aftagende N-
deponering. I de to skovkanter som modtog den mellemste N-deponeringsrate (≤ 23 kg N ha-1
year-
1
) havde skovbundens SOC-indhold tendens til at falde med afstanden fra skovkanten, hvorimod et
årtis eksperimentel N-tilførsel ikke gav en signifikant forøgelse af jordbundens humuslag (H),
hvilket viser en svag tendens for en positiv effekt af N-deponering på SOC-indhold.
De tre skovkanter, som modtog højere N deponeringsrater (> 29 kg N ha-1
år-1
), var N-mættet og
SOC-indholdet i jordbundens øvre lag var enten uændrede eller negativt påvirket af forhøjede N-
deponering. Tilsvarende var skovbundens SOC-indhold uændret efter to årtiers eksperimentel N-
tilførsel. Input af C i form af nåle-litter blev generelt ikke signifikant påvirket af N-deponering i

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skovkantlokaliteterne, men havde tendens til at stige med øget afstand fra skovkanten i to af de N-
mættede lokaliteter. De eksperimentelle N-tilførsler resulterede i reducerede C-input i form af litter
sammenlignet med kontrollen, samtidigt med reduceret træ- og mosvækst. Ved tre skovkanter
tendere biomassen af fin-rødder til at stige med afstand fra skovkanten, hvorimod det
gennemsnitlige indhold af fin-røddernes biomasse, ectomycorrihizal (EM) mycelial produktion og
artssammensætning faldt ubetydeligtover et årti med N-tilførsel sammenlignet med kontrollen,
hvilket indikerer en reduktion af C–input i jorden under forhøjet N-deponering.
Ved to skovkantslokaliteter tendere C-inputtet til skovbunden forårsaget af respiration at falde med
falden C/N-indhold i skovbunden og med afstanden fra skovkanten, hvilket indikerer en positiv
effekt af forhøjet N-deponering på binding af SOC. Tilsvarende blev det fundet at N-beriget litter
inkuberet i litterposer havde en signifikant lavere omsætningsrate i det sene stadie sammenlignet
med kontrol-litter. Dog var skovbundens og jordbundens potentielle respiration generelt upåvirkede
af de eksperimentelle N-tilførsler. Ved ændringer af temperaturen på prøver fra skovbunden fra én
af skovkantslokaliteterne blev det fundet at respirationsrater og temperaturfølsomheden (Q10) var
faldende for tilgængeligt og svært nedbrydeligt SOM med faldende C/N forhold for skovbunden.
Jeg konkluderer, at N-deponeringsrater typiske for landbrugsintensive regioner i det nordvestlige
Europa kan mindske omsætningsraten for SOM og derved have en positiv effekt på binding af SOC
mens C-inputtet til og i jorden mindskes samtidigt, hvilket resulterer i uændrede SOM-indhold i
skovjorden. Endvidere kan forhøjede N-deponering reducere den potentielt positive feedback af
omsætning af SOM på globale atmosfæriske CO2 koncentrationer. Disse resultater har betydning
for modellering af styrken af jordens evne til at binde C i tempererede skove under globale
forandringer.

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List of abbreviations
C: Carbon
CO2: Carbon dioxide
DIN: Dissolved inorganic N
DOC: Dissolved organic C
EM: Ectomycorrhiza
FF: Forest floor
FIA: Flow Injection Analysis
H: Humus layer of the forest floor
KH: Klosterheden (forest edge site)
LF: Litter and fermentation layer of the forest floor
MS: Mineral soil
N: Nitrogen
NUE: Nitrogen use efficiency
PAR: Photosynthetic active radiation
SO: Sønder Omme (forest edge site)
SOC: Soil organic carbon
SOM: Soil organic matter
TF: Thetford (forest edge site)
TG: Thyregod (forest edge site)
VL: Vloethemveld (forest edge site)

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The aim of the project
The overall aim of this PhD project was to unravel the influence of N deposition, a globally
important driving force for environmental change, on SOC processes and stocks in temperate forest
soils. To do this we developed a new experimental platform to study soil C and N interactions based
on short N deposition gradients occurring naturally in forest edges but also used data from a long-
term (20 years) N-addition experiment. We quantified the effects of elevated N deposition on
above- and belowground C inputs to the SOC pool, on SOM turnover, SOC stock and on the
temperature sensitivity of SOM heterotrophic respiration. We focused on the forest floor and top 0-
5 cm mineral soil since the effect of elevated long-term N deposition on soil C is most likely to be
strongest in these layers.
Our specific research questions were:
• How does elevated N deposition affect aboveground C inputs to the soil?
(Papers I and II)
• How does elevated N deposition affect belowground C inputs to the soil?
(Papers I and II)
• How does elevated N deposition affect the decomposition (C outputs) of SOM?
(Papers II and III)
• What is the net effect of elevated N deposition on SOC stocks?
(Papers I and II)
• How does elevated N deposition affect the short-term temperature sensitivity of SOM?
(Paper III)

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General introduction
Nitrogen deposition
During the last century, anthropogenic activities associated with fertilizer applications and fossil
fuel burning resulted in high emissions of reactive N into the atmosphere (Galloway et al., 2004;
Townsend et al., 1996). Consequently, N deposition into forest ecosystems in large parts of Europe
has been increasing from a pre-industrial rate of about 0.5 kg N ha-1
year-1
to 15-50 kg N ha-1
year-1
(Emmett et al., 1998; Fischer et al., 2012).The forests which are especially affected are located
within hundreds of kilometers downwind of large industrial and agricultural areas (Emmett et al.,
1998). In addition, fragmented forests in areas of intensive livestock production can also suffer from
high rates of N deposition (Wuyts et al., 2008a).
Some of the N deposited into forests (up to 50-70% at forests receiving low to moderate N
deposition) can be taken up by the forest canopy (Lindberg et al., 1986; Lovett & Lindberg, 1993)
and be reflected in increasing leaf N concentrations and lower litter C/N ratio (Chappell et al., 1999;
Lovett et al., 2013). Upon leaf senescence, input of N-enriched litterfall to the forest floor together
with N-enriched throughfall would be expected to increase forest floor and soil N concentration and
lower forest floor C/N ratio (Nave et al., 2009). Elevated N deposition into N-limited forest
ecosystems was linked to changes in biodiversity (Gundale et al., 2014), elevated nitrate leaching
(Gundersen et al., 1998), increased susceptibility for insect attack (Pitman et al., 2010), imbalanced
tree nutrition (Fluckiger & Braun, 1998) and soil acidification (De Schrijver et al., 2007b).
Ecosystem partitioning of sequestered C and impacts of N deposition
The close association between the N and C cycles implies that a change in one cycle should be
accompanied by a change in the other. Therefore, N deposition effect on forest SOC stocks is
commonly determined by evaluating effects on above- and belowground C inputs by litterfall and
on C outputs by SOM decomposition as well as on the balance between these two (Figure 1a).

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Figure 1: (a) Schematic representation of N deposition effects on selected C pools and fluxes based on
observations from the N-addition site at Klosterheden (Paper II). Upward and downward arrows indicate C
gains and losses, respectively. (b) A hypothetical response of forest C sequestration in SOM to increasing N
availability (modified from (Gundersen et al., 2006). The dashed line indicates reduction in C sequestration
owing to interacting environmental factors such as increased frost damage or insect infestation.
The response of forest soils to N deposition can theoretically be viewed as a continuum along an N
gradient (Figure 1b). When forests are N limited, addition of N will generally increase SOM
accumulation through positive effects on growth and negative effects on SOM decomposition (Berg
& Matzner, 1997; Fleischer et al., 2013). However, if N deposition continues to increase nitrate
leaching will increase (Dise et al., 1998) meaning less C will be sequestered per unit N added
(reduced nitrogen use efficiency, NUE, Hyvonen et al., 2008). Eventually, chronic high N
deposition may lead to N saturation conditions characterized by altered biogeochemical cycles and
disrupted ecosystem functioning (Aber et al., 1998) implying reduced C sequestration in the long-
run (Magill et al., 2004). However, changes in the N-status of an ecosystem from N-limitation to N-
saturation occur gradually and over a long time period in which the effects mentioned above can
sometimes overlap (Kennedy, 2003). This means that a positive effect of N deposition on SOC

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sequestration by one ecosystem function (e.g. C gains by reduced SOM decomposition) can occur
simultaneously with a negative effect on SOC sequestration by another function (e.g. reduced
litterfall and moss C inputs, Figure 1a). This complexity makes assessment of the N deposition
effect on net forest SOC sequestration difficult especially for forests characterized as having
intermediate N status (Figure 1b) where the effect is not only progressively reduced but may also
shift from positive to negative.
Nitrogen deposition effects on above- and belowground C inputs
As N is the most limiting nutrient for plant growth, low to moderate N deposition to N-limited
forests will typically stimulate tree growth (Thomas et al., 2010) through positive effects on
photosynthesis (Fleischer et al., 2013). Litter input is expected to increase with tree growth (Hansen
et al., 2009) and N deposition was negatively related to needle retention (Kennedy,2003) implying
increased C input by needle litterfall with moderate increase in N deposition. However, at N
deposition rates above 8 kg N ha-1
year-1
the photosynthetic capacity of trees is saturated (Fleischer
et al., 2013). In addition, N fertilization experiments reported reduced NUE of trees with increasing
N additions (Hogberg et al., 2006; Hyvonen et al., 2008) indicating that the positive effects of N on
C sequestration aboveground should be higher for forests receiving low N deposition inputs
(provided that growth is not limited by other factors) and that litterfall may be unchanged or even
decreased under severe N saturation conditions (Aber et al., 1998) owing to nutritional imbalances
and increased susceptibility for insect attack (Fluckiger & Braun, 1998; Kennedy, 2003). Yet, N
addition experiments have shown inconsistent effects on aboveground litter production with
positive as well as no effects reported (Franklin et al., 2003; Magill et al., 2004; Zak et al., 2008).
An N-induced increase in aboveground biomass is typically associated with a simultaneous
reduction in C allocation to roots (Hendricks et al., 2000; Nadelhoffer & Raich, 1992) resulting in
reduced fine-root biomass and their associated mycorrhizal growth (Boxman et al., 1998; Haynes &
Gower, 1995; Treseder, 2004) indicative of reduced belowground C input into the soil. Compared
with the fine-root biomass, less is known about the effects of N deposition on fine-root growth and
turnover. Observations of positive effects of increasing N deposition on tree growth and litter
turnover have led Hendricks et al. (1993) and Nadelhoffer (2000) to hypothesize that fine-root
growth and turnover should exhibit similar responses, i.e. decrease in biomass but at the same time
increase in growth and turnover with increasing N availability. However, removal of N from
throughfall deposition by means of roof was shown to be associated with reduced fine-root growth
(Clemensson-Lindell & Persson, 1995) but also with higher live/dead ratio of fine-roots (Zang et

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al., 2011) which could indicate an increase in fine-root growth in response to reduced N
availability. Yet, the importance of C inputs by fine root and EM turnover to the SOC pool is still
uncertain (Ekblad et al., 2013; Trumbore & Gaudinski, 2003)
Effects on soil C outputs
Decomposition was broadly defined as the physical, chemical and biological processes that
transform organic matter into increasingly stable forms and involve mass loss of organic matter as
CO2 or leachate (Berg & McClaugherty, 2011).
There is ample evidence from respiration and litter decomposition studies that the effect of N
additions on SOM decomposition rate is negative (Berg & Matzner, 1997; Franklin et al., 2003;
Ramirez et al., 2012; Zak et al., 2008, Figure 1a) and may contribute up to a 10% increase in SOC
stocks (Janssens et al., 2010). The inhibiting effect of N addition on litter decomposition was
reflected in higher initial mass loss rates and lower late-stage decomposition rates (so called “limit
values for decomposition”, Berg et al., 1996) for N-enriched litter incubated in litterbags compared
with N-poor litter (Whittinghill et al., 2012). Though some studies related the decrease in SOM
decomposition in response to N deposition to increased chemical resistance of SOM (Fog, 1988;
Nömmik & Vahtras, 1982), there is however evidence that increasing N availability inhibits SOM
decomposition by reducing microbial activity or biomass (Frey et al., 2004) and by shifting
microbial species composition toward a community dominated by species with higher resource
utilization efficiency (Ågren et al., 2001). Moreover, litter in its initial decomposition stage would
be associated with high dominance of fungal and microbial groups specialized in soluble
carbohydrate degradation while cellulose- and lignin-degrading organisms prevail in later
decomposition stages (Dighton, 2007). In addition, high N availability was shown to increase the
activity of microbial cellolytic enzymes (Carreiro et al., 2000) but reduce the activity of lignin-
degrading enzymes (Carreiro et al., 2000; DeForest et al., 2004) which implies a faster turnover of
“young”, labile SOM and a slower turnover of “old” and more recalcitrant SOM under N additions.
Effects on soil C stocks
Depending on its rate and duration, the effect of N deposition on soil C input and output fluxes can
vary from a positive, no effect to a negative effect. For example, faster turnover of “young”, labile
SOM and a slower turnover of “old” and more recalcitrant SOM under N additions may imply an
increasing mass of the forest floor H layer (Berg et al., 2001) and diminishing mass of the LF layer.
These effects on forest floor C stocks are however under the condition that the net C input by tree

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roots and litterfall remain unaffected by N deposition which is unlikely under N-saturated
conditions (Aber et al., 1998). These contrasting effects on specific forest C compartments or fluxes
can make it difficult to determine whether forest soils act as a net C sink or source under increasing
N deposition based on short-term flux measurements. In addition, a recent meta-analysis dealing
with N deposition effects on soil C stocks revealed discrepancies between results from N
fertilization studies and N deposition studies where the former showed an overall stronger positive
response on mineral soil C stocks while C stocks in the forest floor were not affected by both forms
of N input (Nave et al., 2009). This difference is likely due to the intensive nature of N fertilization
experiments, typically applying high quantities of N over relatively short periods (Hasselquist et al.,
2012; Hyvonen et al., 2008) that substantially alter the rates of biogeochemical processes in forest
ecosystems. Therefore, the question still remains whether simulation of N deposition effects on
SOC sequestration by N fertilization truly conforms to effects of the less intense naturally occurring
N deposition.
Why study N deposition effects on forest C sequestration?
Climate change related policies, such as articles 3.3 and 3.4 of the Kyoto Protocol, require countries
to account for the net changes in greenhouse gas emissions by sources and removal by sinks
following activities of land-use, land-use change and forestry. These policies have facilitated a
global effort to estimate the C-sink strength of temperate forests to be used in future C trading
schemes (Sutton et al., 2008). Nitrogen deposition was suggested to be a major driver for forest C
sequestration (Magnani et al., 2007) but its importance is under debate (de Vries et al., 2009; Sutton
et al., 2008), hindering its inclusion in global change models. The debate followed a publication by
Magnani et al. (2007) implying net forest C accumulation of 470 kg C kg-1
N in total deposition (de
Vries et al., 2008) and contradicted considerably lower rates (15-40 and 5-35 kg C kg-1
N deposited
in aboveground forest biomass and soils, respectively) reported by other studies (de Vries et al.,
2006; de Vries et al., 2009; Nadelhoffer et al., 1999).
The importance of reaching reliable estimates of forest C sequestration in response to N
deposition is related to the role of atmospheric N in mitigating climate change through increasing
CO2 uptake by terrestrial ecosystems. High C sequestration potential per kg N indicates high
mitigation potential of atmospheric CO2 concentrations and may justify future elevated N and CO2
emissions. On the other hand, lower estimates may indicate that atmospheric N enhances rather than
mitigates global warming through effects of e.g. N2O on global temperatures (de Vries et al., 2009).
Moreover, high chronic N addition was shown to reduce SOM decomposition (Berg & Matzner,

17
1997; Janssens et al., 2010) thereby lessen global warming. Furthermore, defining thresholds for N
deposition reflecting the change in which forest soils turn from net C sinks to net C sources can
assist in redefining the thresholds for critical loads used widely as a tool in forest conservation.
Nitrogen deposition effects on SOM turnover in a warmer world
As mentioned above, elevated N availability was shown to reduce the rate of SOM decomposition
(Ågren et al., 2001; Knorr et al., 2005; Zak et al., 2008). However, the effect of increasing N
deposition on the temperature sensitivity of SOM decomposition was not addressed directly.
Kinetic principles controlling the temperature sensitivity of SOM (commonly described by Q10 - the
rate of change in a process for a 10°C increase in temperature, Davidson & Janssens, 2006) dictate
that decomposition rates will increase with temperature and that the increase will be greatest under
low temperatures. Moreover, reactants with high activation energies, i.e. more recalcitrant reactants,
should have high temperature sensitivities. Linking these principles to the assumption that most soil
C is stored as relatively stable SOM in temperate and boreal regions of the world, this implies that
increased decomposition of recalcitrant SOM is likely to be the dominant positive feedback process
on atmospheric CO2 concentrations in a warmer world. Therefore, the temperature sensitivity of
SOM is a key issue in the effort to estimate the response of the terrestrial environment to climate
change, but much uncertainty is so far associated with this property of SOM (Conant et al., 2011;
Lloyd & Taylor, 1994; Melillo et al., 2002).
Since decomposition is a complex process including many steps, empirical evidence of the
temperature sensitivity of labile and recalcitrant SOM does not always agree with the last kinetic
principle mentioned above (Conant et al., 2011). Some studies have shown recalcitrant SOM
(defined in terms of age, turn-over time or composition) to be more (Craine et al., 2010; Knorr et
al., 2005), less (Liski et al., 1999) or equally (Fang et al., 2005) sensitive to warming compared
with labile SOM. Most studies of temperature sensitivity dealt with labile SOM with short turnover
rates (Conant et al., 2011) whereas studies on the recalcitrant fraction are less common, perhaps due
to the on-going dilemma as to how to define ‘old’ or ‘recalcitrant’ SOM. A recent review by Conant
et al. (2011) concluded that depolymerisation of complex compounds, microbial enzyme production
and availability of SOM are the three main processes regulating the temperature response of SOM.
Since N availability plays a key role in these processes by regulating tree growth and microbially
mediated C processes it is reasonable to assume that N availability may also affect the temperature
sensitivity of SOM.

18
Forest edges
In terms of the physical environment, forest edges can be defined as a forested zone exhibiting
lateral gradients in microclimatic conditions (such as solar radiation, wind velocity, air and soil
temperatures and relative humidity) owing to the gradual or sudden loss of forest canopy (Saunders
et al., 1991). Forest edges are also characterized by faunal and floral species adapted to the edge
habitat, and species composition at the edge is markedly different from that found in the forest
interior (Honnay et al., 2002). In abrupt forest edges such as those along agricultural fields,
microclimatic gradients can extend up to 240 m into the forest (Chen et al., 1995) though their slope
will depend on the orientation of the edge towards the sun with steeper gradients at south-facing
edges compared with north-facing edges of forests in the northern-hemisphere (Matlack, 1993).
Our aim in Papers I and III was to introduce a new approach to study the effects of long-term N
deposition on soil C under natural deposition regimes as an alternative to the N fertilization
approach (Paper II). We developed a new experimental platform to study the effects of N deposition
on soil C stocks and fluxes at the stand scale. This platform relied on earlier reports of N deposition
gradients decreasing from the edge toward the forest interior (Wuyts et al., 2008a). Since our study
sites were well-established, dense, monoculture conifer forests, we assumed that gradients in
microclimate would be narrow and that any variation in soil C fluxes or stocks would mainly be a
result of changes in the N load. In addition, we assumed that an N deposition gradient in the edge
sites would be reflected by gradients in commonly used indicators for long-term N deposition and
ecosystem N status, such as forest floor and needle litter C/N ratios, soil solution nitrate
concentrations and needle litter N concentrations (Chappell et al., 1999; Dise et al., 1998;
Gundersen et al., 1998) and that these may explain changes in SOC stocks, SOM decomposition
and C inputs to the soil.

19
Methodology
The forest edge sites
The work described in papers I and III was based on N and C data collected along short stand-scale
transects in five homogeneous monoculture forest edges located in north-Western Europe (Figure
2). Two Picea abies sites and one P. sitchensis site in Denmark (Thyregod (TG), Klosterheden
(KH) and Sønder Omme (SO)), one Pinus nigra site in Belgium (Vloethemveld (VL)) and one P.
nigra site in the UK (Thetford (TF)).

20
Figure 2: The location of the forest edge experimental sites.

21
All the stands bordered arable lands dominated by intensive livestock production and have
experienced several decades of elevated N deposition. They were selected to represent high (VL,
TG, TF) to moderate (KH and SO) levels of N deposition (Fischer et al., 2012, Table 1). Facing the
westerly winds and downwind from the arable land, the abrupt stand edges were expected to receive
enhanced levels of N deposition due to increased wind turbulence (edge effect, Beier & Gundersen,
1989). Two forest edges (TG and TF) were subjected to emissions from major local ammonia
sources (animal housing). All stands were located on sandy soils with low base saturation and acid
buffering capacity (pH < 5). The topography was flat in all sites apart from TG which was located
on a gentle west-facing slope (4%). The climate at all sites was temperate with mean annual
temperatures ranging between 7°C and 11°C and mean annual precipitation ranging between 600
and 900 mm. Since the sites differed in several characteristics such as tree species, soil type, land-
use history, age and tree density that could potentially affect the rate of C sequestration above- and
belowground, we focused our analyses on within rather than between stand effects. Potential effects
of other factors such as microclimate, forest floor pH, and previous fertilization on soil C processes
and stocks were also considered, though not in all sites.

22
Table 1: Selected site and stand characteristics: Previous land-use (PLU), mean throughfall N deposition and soil and needle litter C/N at the stand interior (for the
edge sites) or control plot (for the N-addition site), mean annual temperature (MAT), mean annual precipitation (MAP) and reference for further information ;±
values are standard error of means at the stand interior/control. * Latest mean for the combined forest floor LF and H layers. ** For needle litterfall and soil collected
in 2010 and 2008, respectively (unpublished data).
Site name Location Age
(years)
Soil type Tree species PLU N dep.
(kg N ha-1
year-1
)
Needle
litter
C/N
Forest
floor
C/N
Top
mineral
soil
C/N
MAT
(°C)
MAP
(mm)
Reference
Vloethemveld
(VL)
51°08'26''N
3°06'36"E
70 Haplic podzol Pinus nigra ssp. nigra
Arnold
Heathland
grazed by
sheep
33 NA 26±1 24±2 10.4 790 Wuyts et al., 2008a
Thyregod
(TG)
55°54'02''N
9°16'35"E
32 Haplic Arenosol Picea abies (L.) Karst. 2nd
generation
conifers
after
grassland
29 29±2 22±1 20±1 7.5 875 Kjøller et al., 2012
Thetford
(TF)
52°48'77''N
8°38'18"E
35 Ferallic Arenosol Pinus nigra ssp.
Laricio (Poir) Maire
Heathland 21±1 36±1 27±1 22±1 11.3 600 Vanguelova &
Pitman,2009
Klosterheden
(KH)
56°29'32''N
8°19'13"E
89 Podzol (Typic
Haplorthod)
Picea abies (L.) Karst. Heathland 20±5 40±2 31±1 35±1 8 800 Paper III
Sønder
Omme
(SO)
55°49'26''N
8°55'56"E
71 Podzol Picea sitchensis
(Bong.) Carr.
Heathland
with a short
cultivation
phase
19±1 44±1 28±1 31±1 7.3 900 Paper I
N-addition
experiment
56° 29’N
8° 24’E
97 Podzol (Typic
Haplorthod)
Picea abies (L.) Karst. Heathland 55 32** 31.0±0.6*
42** 8 800 Gundersen,1998

23
Experimental design – forest edges
Details on the experimental set up of sampling plots and study periods are provided in Table S1 of
paper I. Two to three transects were established perpendicularly to the forest edge in areas of
homogeneous forest cover not affected by wind-throw damages that are abundant in north-western
Europe. Depending on stand size and homogeneity, each transect consisted of three to eight
sampling plots in fixed distances from the forest edge and with similar tree density and crown
cover. The deposition of air-borne particles to conifer forests was shown to decrease exponentially
with increasing distance from edge (Weathers et al., 2000; Wuyts et al., 2008a). Therefore, to
capture the spatial variability in N and C fluxes close to the edge the distances between plots
increased towards the interior of the forest.
The Klosterheden N-addition experiment
The Klosterheden N-addition experiment started in February 1992 as part of the NITREX project
(Wright et al., 1995) and included monthly N applications (35 kg N ha-1
year-1
as NH4NO3) to a
Norway spruce (Picea abies L. Karst) stand located in the center of the Klosterheden plantation,
near Lemvig, Denmark. Due to financial constraints and a 15
N tracer experiment as the focus, the N
addition treatment was not replicated but surrounded by three control plots (Gundersen &
Rasmussen, 1995, Figure 3). At the sides bordering control plots a plastic trench (25 cm deep)
prevented roots from entering the fertilized plot. The N addition was performed on 500 m2
with a
net area of 15 x 15 m with 22 trees used for measurements. The N addition plus the atmospheric N
deposition resulted in total input of about 50-55 kg N ha-1
year-1
. In a one year pre-treatment period
(1991) variations in N fluxes and other variables among the plots were established for use as
covariates for determining treatment effects (Gundersen & Rasmussen, 1995; Gundersen, 1998).

24
Figure 3: The N-addition site at Klosterheden forest including the experimental design. The yellow plastic
trench separates the fertilized plot (right) from one of the control plots (left).

25
Though the N addition treatment was not replicated in space this experimental design still allowed
for comparisons of treatment effects with multiple control plots in the same stand. Moreover, some
of our investigations were replicated in time (SOC stocks, potential respiration), measured
continuously over long period (tree basal area, litterfall, litterbags, Figure 4) or based on paired
sampling design (potential respiration, litterbags, EM mycelial growth and species composition,
fine-root growth) to increase the probability of identifying treatment effects. Working with pairs
may reduce the influence of spatial variation at 5-7 m scale from e.g. uneven distribution of
thinning residues no longer visible on the site. Paper II summarizes the main results from the N-
addition experiment and aims at indicating a certain direction of change in ecosystem C stocks and
fluxes in response to the N-addition treatment.
Figure 4: Timeline for measurements at the N-addition site at Klosterheden.
Sampling and analysis
Microclimate at the edge sites
Spatial variations in photosynthetic active radiation (PAR) and temperature (̊C) at the soil surface
across the edges were recorded by two replicate probes installed in each plot along one transect at
TF, KH and SO over one- or two-year period.

26
Throughfall
Throughfall N measurements using polyethylene funnels (Figure 5) were taking place at all the edge
sites over one or two years and the results are presented in paper I. On each sampling occasion, the
volume of water from each funnel was recorded, the samples from each plot were mixed and a sub-
sample was analyzed for nitrate-N (NO3
-
– N) and ammonium-N (NH4
+
–N) concentrations by ion
chromatography and Flow Injection Analysis (FIA), respectively. Dissolved inorganic N (DIN; sum
of NO3
-
–N and NH4
+
–N) in throughfall was calculated except in TF where the total N content was
measured due to a large proportion (on average 18%) of organic N in the throughfall. The
throughfall N fluxes were calculated by multiplying N concentrations with water fluxes.
Our one to two years throughfall measurements provide a snapshot of the recent N deposition
gradient in each stand but are likely to underestimate the total atmospheric N input owing to the
canopy uptake of atmospheric N and by neglecting N input by stem flow. Though N input by stem
flow was shown to be less than one percent of the throughfall N input in a Norway spruce stand
(Christiansen et al., 2006), increased wind turbulence at the edge (Saunders et al., 1991) may
increase the proportion of N input by stem flow. Moreover, major differences in throughfall N
deposition gradients between years (Hansen et al., in prep.) and the considerable spatial variability
of throughfall N deposition in relation to nearest stem in coniferous plantations (Beier et al., 1993)
makes our estimates of the N deposition gradients uncertain and necessitated the use of
supplementary indicators for long-term N enrichment .

27
Figure 5: A view from the measurement plot closest to the edge at SO site toward the plots at the interior.
Each plot consisted of three throughfall funnels connected to a bottle located in a PVC cylinder underground
and three circular litter traps.
Foliar litter
Aboveground C input to the soil is typically assessed by measurements of litterfall mass captured in
litter traps or fiber sheets installed on poles aboveground or laid on the soil surface, respectively.
We collected litterfall during one to two consecutive years at each of the edge sites and for a longer
period in the N-addition site by circular litter traps (Ø either 31 or 50 cm) consisting of a
polyethylene net mounted on wooden poles about 1.5 m above the ground (Figure 5). The collected
litterfall was dried at 55 °C, sorted into needle and non-needle (mainly branches and cones)
fractions and the weight of each fraction recorded and pooled to annual fluxes. Each fraction was
ground finely and analyzed for C and N concentrations by the DUMAS method (Matejovic, 1993).
The annual needle litter element flux (g m-2
year-1
) for each plot was then estimated by multiplying
the element concentrations with the annual litterfall dry mass. Due to the relatively short sampling

28
period and the high spatial variation in coarse woody litterfall (cones, branches and twigs)
compared with needles litterfall we included only the latter in our estimations of litterfall C input.
However, C input by coarse woody debris can count up to 73% of the aboveground C input and up
to 54% of the accumulated organic matter in northern coniferous forests (Laiho & Prescott, 2004).
Estimations of C input by litterfall are usually associated with high levels of uncertainty owing
to potential effects of microclimate as well as insect and fungal infestations on the litterfall pattern
(Finer, 1996; Kennedy, 2003; Pitman et al., 2010). These factors can vary and interact during or
prior to the measurement period and can strongly affect C and nutrient flux into the soil. In addition,
the timing of litterfall varies among tree species. For instance, Norway spruce is characterized by
having an even litterfall throughout the year while Scots pine sheds most of its litterfall between
August and October (Finer, 1996; Mälkönen, 1974). Therefore, the litterfall estimates in Paper I
were based on repeated measurements during one or two full years and were compared, whenever
possible, with data from previous measurements.
Forest floor and mineral soil sampling
Assessing forest floor C stocks was done in a similar way in the edges and in the N-addition
experiment (Papers I and II) by sampling of the forest floor using a 25 cm x 25 cm wooden frame
(Vesterdal & Raulund-Rasmussen, 1998, Figure 6a). The forest floor samples (15 replicates at each
distance) used for the temperature incubation experiment (Paper III) were collected using a soil
corer (Ø4.2 cm) inserted perpendicular to the forest floor surface. The samples used in papers II and
III were separated by knife into the LF layer which consisted of recognizable needles and bits of
needles and to the humus (H) layer consisted of organic material of an unrecognizable origin.
Sampling of the mineral soil (0-5 cm depth) at the edges was done by a similar soil corer as above
used to collect 16 replicates (4 inside each frame) of the exposed mineral soil (Figure 6b) which
were pooled to one sample per plot.
Our aim was to identify long-term effects of N deposition on SOC stocks and fluxes and the
bulked samples were therefore sieved through (4 mm x 4 mm) to remove coarse material, living
roots and stones and dried at 55 °C to obtain the dry weight. The dried samples were weighed,
finely ground and then analyzed for C and N concentrations by dry combustion. Soil C stocks (Mg
C ha-1
) were calculated separately for each layer by multiplying the C concentrations by the soil dry
mass of the layer. Soil organic matter C/N ratio was calculated using the C and N concentrations.
Our forest floor and soil fractionation method was based solely on visual distinction between
soil layers and on soil sieving. However, the fine SOM fractions of both layers consist of

29
compounds of different age and various levels of stability (Schulze et al., 2009). At present, there is
no consensus as to which soil fractionation method is most suitable to characterize SOM pools of
homogeneous stability (von Lutzow et al., 2007). Therefore, the definitions of ‘labile’ vs
‘recalcitrance’ used in this study should be used in a relative rather than absolute sense.
Figure 6: Sampling of forest floor in a 25 cm X 25 cm wooden frame (a) was followed by sampling of the 0-
5 cm mineral soil by coring (b).
Fine roots
We estimated the effects of N deposition on C inputs belowground by assessing the relative changes
in fine-root biomass, production and turnover. Of these three indicators, fine-root turnover, defined
as the replacement of a particular standing stock (Lauenroth & Gill, 2003) can better represent a net
change in belowground C sequestration by fine-roots as it integrates valuations of biomass and
growth, hence it requires more data. Assessing changes in belowground C inputs using fine-root
biomass as a proxy can be subjected to bias owing to spatially differing rooting patterns between
tree species (Hansson et al., 2013). This means that estimates of belowground C input based on
sampling at the top soil may be underestimated for species such as Scots pine that are characterized
by relatively larger root biomass at deep soil layers.
Fine-root biomass was estimated at four edge sites while fine-root biomass, growth and turnover
were estimated at the N-addition site. The sampling of biomass at all sites was done using a soil

30
corer (Ø4.2 cm) inserted perpendicularly to the soil surface to a depth of 20 cm following
separation of the sampled material into forest floor and mineral soil layers. Live fine tree roots (Ø<2
mm and length >1 cm) were picked by hand based on color and physical appearance, rinsed with
distilled water and dried at 55 °C. The fine-root biomass (g m-2
) for each layer was calculated by
dividing the root dry weight by the sampled area.
Annual root growth (as g m-2
year-1
) was estimated using ingrowth cores placed in the holes left
after the root biomass samples. The ingrowth cores were made of a nylon mesh tube (mesh size 1
mm) and filled with root free soil from a nearby soil pit, first with mineral soil up to the mineral soil
surface and then with LFH material sieved (4 x 4 mm) and mixed before use. During filling the soil
was compressed to obtain approximately the same bulk density as in the bulk soil. After one year
the ingrowth cores were retrieved, the surrounding roots and soil removed and the forest floor was
separated from the mineral soil. Roots were sorted and treated as above. Fine-root turnover rate was
estimated by dividing the annual root growth by the biomass (Brunner et al., 2013). Nitrogen
deposition effects on belowground C inputs were also estimated by assessing N-treatment effects on
EM mycelial production and species composition (Paper II) using the ingrowth mesh bag technique
(Wallander et al., 2001; Wallander et al., 2013). The ingrowth core method was criticized to
overestimate fine root production since roots can proliferate due to the lack of competition for
nutrients in the cores, higher nutrient availability following soil disturbance, the soil coring and due
to changes in the bulk density of the soil (Joslin & Wolfe, 1999). In addition, the importance of N-
mediated interactions between fine-root biomass and root production and turnover to belowground
C sequestration are poorly understood (Nadelhoffer, 2000).
Nitrate leaching
Elevated nitrate leaching below the root zone reflects a situation where the N input to an ecosystem
exceeds the ecosystem’s N retention capacity and was shown to increase with N deposition (Dise &
Wright, 1995). Since nitrate leaching fluxes were not available for all the sites we used the soil
NO3
-
– N concentrations below the root zone as an indicator for N leaching and N status of the site.
Soil NO3
-
– N concentrations below the root zone were measured in VL, TG, TF and the N-
addition site by extracting soil water using suction lysimeters inserted below the root zone. At KH
and SO, soil solution NO3
-
– N concentrations were measured by KCl extraction of mineral soil
material sampled in the dormant season using a soil corer (Ø3 cm), a method that yield similar
results for NO3
-
– N as soil water samples (Djurhuus & Jacobsen, 1995) but with a low temporal

31
resolution. Field NO3
-
– N concentrations (mg NO3
-
– N l-1
) were estimated using the filtered solution
concentrations and the field soil water content.
SOM turnover
In conifer forests on a flat landscape, the main pathway by which C can be lost from the soil is
through microbial decomposition of SOM that can be measured as heterotrophic respiration or by
estimating the mass loss of litter. Bulk soil respiration includes both autotrophic and heterotrophic
components that can be measured separately in the field or in highly controlled laboratory
microcosms (as potential respiration).
Measurements of SOM heterotrophic respiration were done in all the papers and included
incubation of soil samples in a respirometer at constant abiotic conditions for various time periods.
This relatively simple method allowed us to isolate the effects of N deposition on heterotrophic
microbial activity and C outputs from the effects of other environmental factors, such as soil water
content and temperature that may be variable in small scales and strongly affect soil microbial
activity (Reichstein et al., 2005).
The incubation experiments in the thesis were conducted by placing soil and forest floor
samples in microcosms (polyethylene vessel) equipped with a small conductivity cell attached
under the lid (Figure 7a). The conductivity cell contained a KOH solution and two platinum
electrodes for recording the decrease in the electrical conductivity of the solution capturing the
respired CO2. The change in the electrical conductivity is proportional to the soil CO2 efflux
(Nordgren, 1988), which was obtained as mg CO2 hour-1
and was recalculated to a mean respiration
rate as mg CO2-C g soil C-1
h-1
(assuming 50% C in SOM) to evaluate the reactivity of the SOM.
The whole experimental unit was placed in a water bath filled with distilled water and connected to
both a cooler and a heater (Figure 7b).

32
Figure 7: Incubation of soil samples in a microcosm equipped with a conductivity cell under the lid (a) and
placed in a temperature-regulated water bath (b).
According to Michaelis-Menten kinetics, the CO2 efflux from the incubated samples was expected
to exhibit an initial linear relationship with the incubation time up to the point where labile C
becomes a limiting factor for microbial activity. Thereafter a decrease in the rate of CO2 efflux
would be expected (Davidson & Janssens, 2006) signifying that respiration is increasingly
controlled by the recalcitrant SOM pool. Therefore, studies assessing the decomposition of SOM
pools with different turnover rates typically involve incubation measurements for long periods (e.g.
few months). We have not observed such a decline in a 17-week soil incubation experiment during
2007 (Paper II) and due to financial and technical constraints we therefore conducted the other two
incubation essays for periods of two to three weeks (papers II and III).
Nitrogen deposition effects on SOM decomposition was assessed by incubating needle litter in
litterbags over a five-year period (Paper II). The litterbags were made of a nylon mesh (size 0.5 mm
x 1 mm) with a size of approx. 10 cm x 15 cm and filled with 0.7 g air-dried needle litter. A subset
of samples was dried at 55 ºC for conversion of air dried to oven dried weight. The litterbags were
incubated at randomly chosen subplots (Figure 8) and retrieved at sequential sampling over a 60
months period. Later, the samples were dried to constant weight at 55 ºC and the mass loss for each
incubation period was calculated as percentage of the initial dry weight. Based on the mass loss data
we estimated the limit values for SOM decomposition, as % accumulated mass loss, using an
asymptotic model (Berg & Ekbohm, 1991; Berg & Laskowski, 2006). The limit-values concept for
plant litter decomposition suggests that a certain fraction of litter is not degraded but is being stored
as recalcitrant compounds in soil organic matter (Berg & Ekbohm, 1991). One shortcoming of the

33
litterbag method is the exclusion of soil macrofauna from the litterbags which may slow down SOM
decomposition rates (Bradford et al., 2002) by reducing the litter surface area accessible for soil
microorganisms through litter dissection and mixing with older SOM material. However, it is
unlikely to be of high importance for decomposition in our site owing to the rather low macro-
faunal activity. In fact, reduced water penetration into the litterbag, i.e. lower water content of the
incubated material compared with the surroundings, is likely to be of greater importance for litter
decomposition in our site and may lead to underestimations of “limit values”.
Figure 8: The 2001-2006 litter decomposition study at the N-addition site at Klosterheden.
Temperature sensitivity
The temperature sensitivity of SOM heterotrophic respiration is commonly described by the Q10
expression. Estimates of Q10 can be based on the actual measured response of a given process to a
change in temperature or on a variety of models (Sierra, 2012). The commonly used Arrhenius
equation (Arrhenius, 1889) allows for an estimation of Q10 for every part of the respiration response
model. For instance, if we incubated soils at 5, 10, 15 and 20°C we could calculate Q10 for the 5-15

34
and 10-20 °C temperature ranges. Therefore, it can better illustrate variations in temperature
sensitivities between cold and warm regions (Kirschbaum, 1995). In paper III we were however
interested in the mean short-term response of Q10 to increasing N availability. For this reason and
for the sake of simplicity an exponential function of the form Q10 = e10*α
(supplementary
information, Paper III) was preferred as it integrates the response for the four temperature
treatments to a single Q10 value.
Results
The results below are presented in papers I-III and are therefore only summarized here.
Forest edge effects on N deposition and N availability
Nitrogen deposition gradients decreasing from the edge towards the stand interior were identified at
all the experimental stands but differed in shape and length into the forest interior (Figure 9). The
mean N deposition estimates at the stand interior were 32.5, 28.5, 20.5, 20.1, and 18.6 kg N ha-1
year-1
for VL, TG, TF, KH and SO, respectively (Table 1).
Figure 9: The edge effect on throughfall N deposition along transects expressed relative to the N deposition
at the interior of each forest stand.

35
Depending on the stand, one or several indicators for long-term N availability showed N enrichment
near the edges (Table 2, Paper I) and revealed ‘distance from the edge’ as a reasonable proxy for the
stand-scale N availability gradients.
Table 2: Effects of increasing distance to the forest edge (p-values) and direction of change
(positive/negative) in C and N parameters based on regression analyses. The bold and underlined numbers
show significant and marginally significant relationships, respectively. The up- and downward arrows
indicate whether the relationship was positive or negative, respectively. NA= not measured/available.
Parameter type Parameter Effects related to distance from edge (m)
VL TG TF KH SO
N enrichment Throughfall N (kg ha-1
) < 0.03 ↓ < 0.06 ↓ < 0.001 ↓ < 0.01 ↓ 0.10 ↓
N enrichment Needle litter C/N NA 0.45 0.06 ↓ < 0.05 ↑ 0.06 ↑
N enrichment Needle litter [N] NA 0.37 0.14 0.07 ↓ 0.15
N enrichment Forest floor C/N 0.34 0.26 < 0.001 ↑ < 0.01↑ 0.15
N enrichment Mineral soil C/N 0.69 0.97 < 0.02 ↑ 0.75 0.91
N enrichment Soil solution NO3
-
– N (mg l-1
) 0.12 < 0.05 ↓ 0.18 0.19 < 0.05 ↓
C-input Needle litterfall C (g m-2
yr-1
) NA 0.30 < 0.003 ↑ 0.92 0.93
C-input Forest floor fine-roots (g m-2
) NA < 0.05 ↑ 0.17 0.09 ↑ < 0.001 ↑
C-input Mineral soil fine-roots (g m-2
) NA 0.60 < 0.01 ↓ 0.34 < 0.004 ↑
C-output Respiration NA 0.11 NA < 0.05 ↑ NA
C-stock Forest floor C (t ha-1
) 0.30 0.18 0.41 < 0.01 ↓ <0.04 ↓
C-stock Mineral soil C (t ha-1
) 0.26 0.19 0.78 0.67 0.85
A more detailed soil sampling at KH showed clearly that the edge effect on long term N deposition
was reflected in the C/N ratio of the LF and H forest floor layers that increased significantly with
increasing distance from the edge (Figure 10, Paper III).

36
Figure 10: C/N ratio for the Litter-Fermentation (LF) and Humus (H) layers along two transects from the
edge to the interior (n=7) at Klosterheden. Each point represents a bulked sample based on 15 individual
sampling points around each distance. Continuous and dashed lines are linear regressions for the LF and H
layer, respectively.
The N-treatment and edge effects on N deposition, needle-litter, forest floor and mineral soil C/N
ratios as well as soil solution nitrate concentrations are described in papers I to III. The major
findings were:
• Mean annual N deposition at the edge stands was highest (≥29 kg N ha-1
year-1
) in VL, TG
and TF owing to high regional N emissions (VL) or close proximity to local ammonia
sources (TG & TF) and lowest (≤ 23 kg N ha-1
year-1
) at KH and SO.
• The N deposition gradient reached 180 m into the forest in TF and up to 60-80 m into the
forest in the other sites (Figure 9).
• Needle litter C/N ratios had tendency to increase towards the forest interior at KH and SO
(Table 2).
• Forest floor C/N ratios at the edges were generally lower compared with the stands interior.
However, significant relationships between the forest floor C/N and the distance from the
edge were only observed for TF (p<0.001) and KH (p<0.01, Table 2).

37
• At the N-addition site, the C/N ratio of the LF and H layers were decreased by the N
treatment and C/N ratio of the newly sequestered SOM (2001 to 2013) decreased from 31.8
on the control to 25.0 on the N-plot (Table 4).
• Soil solution nitrate concentrations tended to decrease with increasing distance from the
edge at VL, TG and TF reflecting higher N enrichment at the edges and N-saturated
conditions.
• Low soil solution nitrate concentrations at the KH and SO edges and the N-addition site
indicate high N retention although nitrate leaching has been increasing slightly (up to about
3 kg NO3
-
-N ha-1
year-1
) during the last decade for the latter site.
Nitrogen deposition effects on aboveground C inputs
Our measurements of litterfall as well as tree and moss growth returned the following main results:
• At the edges, annual needle litterfall C inputs were significantly positively related to the
distance from the edge only at TF (Table 2) and tended to increase with distance from edge
at TG (Figure 11a, Paper I).
• At the N-addition site, needle litterfall mass was about 30% higher in the N-plot compared
with control during the pre-treatment year 1991 (Figure 11b, Paper II). However, the ratio of
N:Control litterfall was reduced under the treatment and was about 1 during 2002 and 2010
indicating reduced C input rates under N additions.
• At the N-addition site, cumulative tree basal area in the N-plot was consistently reduced
since 2002 compared with control indicative of growing N stress on tree growth (Paper II).
• At the N-addition site, C input by moss, the dominant understory vegetation, was reduced
from 9.0 to 2 g C m-2
following 20 years of N additions.

38
Figure 11: Nitrogen deposition effects on aboveground C inputs to the soil as indicated by (a) needle
litterfall mean annual C fluxes (g C m-2
yr-1
) as a function of distance from the forest edge in four edge stands
and (b) the yearly ratio in needle litterfall flux as measured in the N and control plots at the N-addition site.
Values for year 1991 are baseline estimates prior to the N addition treatment. The vertical lines in a indicate
standard error of mean.
Nitrogen deposition effects on belowground C input by roots and EM
Changes in C input belowground in response to N deposition were assessed by identifying changes
in fine root biomass, growth and turnover as well as changes in EM growth and species
composition. The results are described in Papers I and II.
The main findings were:
• At the edge sites, fine-root biomass tended to increase with increasing distance from the
edge indicating reduced belowground C input with increasing N deposition (Figure 12).
• At the N-addition site, total fine root biomass, fine root growth, root turnover and EM
mycelial production did not differ significantly between the plots following cumulative N
additions of 490, 525 and 595 kg ha-1
year-1
, respectively (Table 3, Paper II).

39
• At the N-addition site, means of both fine root biomass and mycelial production were 13%
and 30% lower in the N-plot compared with control, respectively (Table 3, Paper II).
• Ectomycorrhizal species richness had decreased by 2009, mostly due to the disappearance of
rare species from the N-plot.
Figure 12: Forest floor fine root biomass (g m-2
) as a function of distance from the edge in four stands. Root
biomass in VL was not measured.
Table 3: Means (±SE) of fine (Ø<2 mm) root biomass (n=10), fine root production (n=10), root turnover
(production:biomass) and EM mycelial production (n=36) as measured in the N and control plots at
Klosterheden. Root biomass and production are given for the combined forest floor and 10 cm mineral soil.
Parameter Sampling
year
Cumulative N
added
1992-2013
(kg N ha-1
)
Treatment Treatment
effect
test
Control N P
Fine root biomass (g m-2
) 2007 490 597±80 519±39 0.2
Fine root production
(g m-2
year-1
)
2007-8 525 177±21 175±18 0.7
Fine root turnover (year-1
) 2008 525 0.30±0.1 0.34±0.1 0.3
EM growth (g m-2
year-1
) 2010 595 10±2 7±1 0.3

40
Nitrogen deposition effects on SOM turnover
The effect of N deposition on SOM decomposition at the edges was assessed by lab-based
respiration essays of soil samples from the KH and TG edges (Figure 13, Papers I and III). At the
N-addition site, N-treatments effects on SOM decomposition were assessed by two respiration
essays (Figure 14), litterbag decomposition studies (Figure 15) and by in-situ respiration
measurements.
The major findings were:
• At the edge sites, forest floor SOM respiration rates tended to decrease with increasing N
availability exemplified by decreasing distance from the edge (Figure 13) or by decreasing
C/N ratio of the samples.
Figure 13: Forest floor respiration rates (µg CO2-C g-1
C hour-1
) at 15o
C as a function of distance from the
forest edge at the TG and KH edges. At KH, the LF and H layers were treated separately.
At the N-addition site:
• Mean potential respiration of the LFand H forest floor layers and the top mineral soil were
not significantly affected by the N treatment (Figure 14). However, the mean respiration
rates in 2003 were higher for the N-treated LF layer and lower for the N-treated H layer
compared with control.

41
• Reciprocal incubation of litter in litterbags revealed that effects of litter quality, i.e.
endogenous N, were more important in determining the final course of decomposition than
effects of exogenous N (Figure 15a and b).
• Nitrogen-rich litter incubated in litterbags had significantly higher mass loss rates in the
early stages of decomposition but significantly lower mass loss rates at the end of the
incubation period compared with control litter (Figure 15b).
• The respiration rate of the bulk soil during 2002 was not significantly affected by the N
treatment.
Figure 14: Mean potential heterotrophic respiration rates of the LF and H forest floor horizons and
top 10 cm of the mineral soil (Min) horizon in the N-plot (N) and control plot (Con) during 17 weeks
(n=6) and 3 weeks (n=4) of incubation in 2003 and 2013, respectively.

42
Figure 15: Accumulated mass loss (AML %) for Norway spruce (Picea Abies) needle-litter a: effect of
exogenous N by incubation in the N-plot and control plot (Con) during 25 and 60 months in the 1992-1994
and 2001-2006 litterbag studies, respectively. b: effect of endogenous N on mass loss rates in the 2001-2006
litterbag study. Error bars indicate standard error of the mean (n=15 for first study; n=30 for second study).
Significance codes: ‘***’ (P<0.001), ‘**’ (P<0.01), and ‘*’ (P<0.05) relate only to the second study.
Nitrogen deposition effects on SOC stocks
Nitrogen deposition (measured over 1-2 years) turned out to be a poor indicator for changes in
forest floor C/N ratio and C stocks within stands (Figure 16a and b, Appendix in Paper I). We thus
assessed the effects of N deposition on SOC stocks using indicators for N availability, such as C/N
ratios in needle litter, forest floor and mineral soil, reflecting several decades of elevated deposition
as well as distance from the edge.

43
Figure 16: Forest floor C stocks (a) and forest floor C/N ratio (b) as a function of nitrogen deposition in the
five stands. The vertical lines mark the standard error of mean.
The main findings for the edge sites were:
• Forest floor C stocks were significantly negatively related to distance from the edge only at
SO and KH (Table 2, Figure 17a), the sites receiving the lowest mean annual N deposition
(Table 1).
• Forest floor C stocks were significantly positively related to forest floor C/N ratios at VL
(p=0.002) and a positive relationship was marginally significant (p=0.07) at TG indicating
lower forest floor C stocks under high N availability at N-saturated sites (Figure 17b).
• Mineral soil C stock was not related to distance from edge in either of the stands (Table 2)
but was significantly positively related to mineral soil C/N ratio at TG (p=0.004) and TF
(p<0.001) indicating lower mineral soil C stocks under high N availability at N-saturated
sites.

44
• Relative to the estimates at the stand interior, increasing N deposition was generally
associated with higher forest floor C stocks across stands but no trends were observed within
stands (Paper I).
• Relative to the estimates at the stand interior, increasing N input within each stand was
overall associated with both increased and decreased forest floor C stocks (Paper I).
Figure 17: Forest floor C stocks as a function of (a) distance from the forest edge and (b) C/N ratio
in five stands. The vertical lines indicate standard errors of means.
At the N-addition site, the effects of the N-treatment on the forest floor C stocks was assessed
separately for the LF and H layers in 2002 and 2013 (Table 4, Paper II).
At the N-addition site:
• Total forest floor C and N stocks were similar for control and N-plot following accumulated
N additions of 315 (by 2002) and 700 kg ha-1
(by 2013, Table 4).

45
• In the period 2001-2013, total C and N stocks increased in both treatments and mainly in the
H-layer. The increase in total forest floor C stocks were 2.3 and 1.7 kg C m-2
(192 and 146 g
C m-2
yr-1
) for the control and the N-plot, respectively, whereas the increase in N stocks
were 71 and 69 g N m-2
(6.0 and 5.8 g N m-2
yr-1
) for the control and the N-plot,
respectively.
• In 2001, the C stocks of the LF-layer was significantly lower while the H-layer more
abundant on the N-plot than on the control. However, this had changed in 2013 where the H-
layer was more abundant on the control than on the N-plot.
Table 4: Mean (±SE) C and N stocks as well as C/N ratio of the LF and H forest floor horizons in the N and
control plots at Klosterheden as measured following 9 and 21 years of N additions (n=10). C/N SOM is the
C/N ratio of the SOM sequestered from 2001-2013. Statistically significant and marginally significant values
are bold and underlined, respectively.
Treatment Layer Carbon (kg C m-2
) Nitrogen (g N m-2
) C/N C/N SOM
2001 2013 change Sign. 2001 2013 change Sign. 2001 2013 Sign.
Control
LF fine 3.0±0.1 3.0±0.2 0.0 0.9 96±4 98±6 1 0.77 31.0±0.3 30.4±0.6 0.2
H fine 2.1±0.2 3.4±0.4 1.2 0.02 62±5 107±20 45 0.02 34.8±0.2 31.5±1 <0.001 26.9
Total fine 5.1±0.1 6.3±0.2 1.2 0.04 158±5 205±10 47 0.02 32.5±0.5 31.0±0.6 <0.001 25.9
Total LFH 5.5 7.8 2.3 164 235 71 33.9 33.3 31.8
N-plot
LF fine 2.7±0.2 3.0±0.2 0.3 0.33 91±6 113±9 22 0.07 29.5±0.5 26.9±0.5 <0.01 15.9
H fine 2.3±0.2 2.9±0.2 0.6 0.11 66±5 92±6 25 0.01 35.1±0.2 31.8±0.8 <0.001 23.1
Total fine 5.0±0.1 5.9±0.2 0.9 0.06 157±5 204±6 47 <0.01 31.9±0.7 29.1±0.7 <0.01 19.8
Total LFH 5.4 7.1 1.7 163 232 69 33.1 30.7 25.0
Nitrogen deposition effects on Q10 of soil respiration
The effect of long-term N deposition on heterotrophic respiration under increasing temperature was
assessed separately for the LF (labile) and H (recalcitrant) forest floor fractions at Klosterheden
edge and the results are presented in Paper III. Here we only show the effect of increasing N
deposition, exemplified by decreasing C/N ratio, on the temperature sensitivity (Q10) of SOM
respiration for these two forest floor layers (Figure 18). To strengthen the observed relationship
between Q10 and the C/N ratio of forest floor layers we included in our regression analysis data from

46
the recent study by Erhagen et al. (2013) on heterotrophic activity in Swedish forest sites using
similar methods.
The main findings were:
• The C/N ratio of the LF and H forest floor layers gradually and significantly increased with
increasing distance from the edge (Figure 10).
• At the 15°C temperature treatment respiration rates of the LF and H layers were
significantly positively related to their C/N ratio.
• The respiration of the LF materials with C/N < 25 (produced under high N availability)
responded less to temperature increase compared to materials with C/N > 25 (p < 0.02).
• Q10 of soil respiration for samples taken from the LF layer at KH decreased significantly (p
= 0.04) by 16% with decreasing SOM C/N ratio while Q10 of the H layer respiration
exhibited a marginal significant decrease (p = 0.10) by 28% with decreasing SOM C/N
ratio.
• Combining the data from the Swedish sites with the KH edge data improved the
relationships between Q10 and SOM C/N ratio for the two layers which appear valid for the
whole C/N ratio range found in organic layers of boreal-temperate coniferous forest (Figure
18).

47
Figure 18: Relationship between the temperature sensitivity (Q10) of SOM respiration and the C/N ratio of
SOM for the litter-fermentation (LF, white circles) and humus (H, white squares) layers collected from the
Klosterheden edge and for data from Swedish forest sites used in Erhagen et al. (2013) including the 0-3 cm
(LF, black circles) and 5-8 cm (H, black squares) forest floor layers. Continous line indicate regression for
the LF layer while dashed line indicate regression for the H layer.
Summary discussion
The aim of this PhD study was to assess the effects of N deposition on SOC processes and stocks in
the top soils of temperate conifer forest using data from natural N-deposition gradients at forest
edges and data from a long-term (20 years) N addition experiment at Klosterheden forest. The text
below addresses the research questions mentioned in the introduction by linking the results from all
the three papers together. It includes critical reflections on the assumptions made and methods used
in the individual papers and ends up with recommendations for policy makers and future research
focus.
Nitrogen deposition and N availability
High spatial variation associated with short-term throughfall sampling may, in part, be the reason
why throughfall N deposition was a poor indicator for changes in forest floor C/N ratio and C
stocks (Figure 16). Therefore, the C/N ratios in needle litter, forest floor and mineral soil, and soil
solution NO3
-
– N concentrations were included as indicators for long-term N enrichment. The
applicability of these indicators for N availability is further discussed in Paper I. Depending on the

48
stand, one or several of these indicators of N availability showed N enrichment near the edges
(Table 2) and revealed ‘distance from the edge’ as a reasonable proxy for the stand-scale N
availability gradients.
Relying on the work of Dise et al. (1998) that assessed the risk of nitrate leaching from
European coniferous forests based on throughfall N input and forest floor C/N ratio I made a
general distinction between the experimental sites or duration of the N-addition experiment based
on their ecosystem N-status (Table 5). Of the edge sites, KH and SO received intermediate N
deposition inputs (≤ 23 kg N ha-1
year-1
), had highest needle, forest floor and mineral soil C/N ratios
and lowest soil solution nitrate concentrations (Table 1 and Appendix in Paper I). Similarly, the first
decade of N applications at the N-addition site did not affect the forest floor C/N ratio (mean 32 for
the LFH layer) and the trees remained unaffected. I therefore regard the ecosystem N-status of these
edges and first decade of the N-treatment as intermediate-N sites. The VL, TG and TF edges
received higher N deposition rates (≥ 29 kg N ha-1
year-1
), had lower needle, forest floor and
mineral soil C/N ratios and higher soil solution nitrate concentrations. Similarly, the C/N ratio of
the fine forest floor at the N-addition site has decreased from 32 to 29 during the following two
decades of N-treatment (Table 4) while soil solution nitrate leaching had been slightly increasing.
Considering the critical load for forest N input as 10-20 kg N ha-1
year-1
(Hicks et al., 2008), high
risk for nitrate leaching in these sites (Dise et al., 1998) and negative effects on tree growth and
foliar litter inputs (see below), I therefore regard VL, TG, TF and the N-addition site after 2003 as
N-saturated sites.
Table 5: Annual mean estimates of selected indicators for forest N-status. Estimates for the edge sites are
based on annual mean along the transects.
Parameter Site
VL TG TF N-addition
2003-2013
KH SO N-addition
1992-2002
N-saturated intermediate-N
Throughfall N
(kg N ha-1
year-1
)
47 35 29 50 23 20 50
Needle litter C/N NA 27 36 32 37 42 35
Fine LFH C/N 25 22 26 29 28 27 32
MS C/N 24 20 22 42 33 29 42
NO3
-
–N (mg l-1
) 61 14 32 1-2 <1 <1 1

49
How does elevated N deposition affect aboveground C inputs into the soil?
The direct measurements of C input by needle litterfall exhibited either no response or a moderate
negative response to increased N deposition depending on the N status of the site. At the
intermediate-N sites (KH and SO), needle litterfall was not related to the distance from the edge
indicating no response to N deposition.
At the N-saturated edges (TG and TF) as well as at the N-addition site needle-litterfall seemed to be
reduced with increasing or continuous N load (Figure 11). For the latter site, tree basal area
increment at the N-plot has been consistently decreasing from 2002 onwards compared with control
signifying that litterfall should also be reduced (Hansen et al., 2009) and concomitant with
increasing, yet low, nitrate leaching rates. These observations confirm our assumption that C input
by litterfall is likely to decrease under high N deposition or severe N saturation conditions (Aber et
al., 1998).
How does elevated N deposition affect belowground C inputs into the soil?
The increasing amounts of fine-root biomass with increasing distance from the edge at TG, KH and
SO and the somewhat reduced fine root biomass at the N-plot during 2007 indicated decreasing
belowground C inputs to the SOC pool under increasing N deposition. These trends, which are in
line with previous reports of reduced fine root biomass following experimental N additions (Aber et
al., 1985; Boxman et al., 1998; Magill et al., 2004) were attributed to reduced C allocation to the
roots system (Hendricks et al., 2000; Nadelhoffer & Raich, 1992). Considering that mycorrhizal
growth is based on C supply from the host tree, this may explain the lower mean EM mycelial
production observed in the N-plot in 2010. Though we did not investigate the effects of N
deposition on EM growth in the edges, an earlier study at the TG edge (Kjøller et al., 2012) found
reduced EM mycelial production and root-tip abundance with increasing N deposition confirming
the general assumption of negative effects of N availability on mycorrhizal abundance (Boxman et
al., 1998; Treseder, 2004).
The results above provide indirect evidence for negative effects of increased N deposition on
belowground C inputs to the soil. However, they do not provide information on the magnitude of
the response since fine-root biomass do not represent a net C input and our estimates were
associated with high uncertainties. Nevertheless, since the declines in C inputs were observed under
intermediate to high ecosystem N status we would expect similar response also in the N-saturated
sites TF and VL (Aber et al., 1998; Magill et al., 2004). However, such an effect was not evident at
the TF edge since the relatively dry conditions in the topsoil probably hampered root growth and

50
thus were more important for fine root biomass distribution than N deposition. However, pines were
also shown to have lower fine root biomass in the top soil compared with spruce (Hansson et al.,
2013) meaning that detecting an effect of N deposition on fine-root biomass at TF may require
sampling to deeper depth, especially under high N deposition load.
Negative effects of increased N availability on the distribution of fine-roots and mycorrhizal
fungi may positively contribute to elevated nitrate leaching below the root zone (Nadelhoffer,
2000), but this potential effect was not assessed in this study.
In the N-addition site, the lower mean fine-root biomass but similar fine-root growth in the N-
plot during 2007 resulted in higher mean fine-root turnover. Increased fine-root turnover may
indicate that trees allocate more C to the production of new roots but spend relatively less C on root
maintenance and production of protective compounds. This suggests that N-rich roots may
decompose faster compared with N-poor roots (Nadelhoffer, 2000) resembling N effects on
aboveground litter decomposition discussed below. However, little is known about the
consequences of increased turnover of fine-roots on SOC sequestration in forests (Nadelhoffer,
2000; Trumbore & Gaudinski, 2003).
How does elevated N deposition affect the decomposition (C outputs) of SOM?
The general hypothesis of reduced SOM decomposition under elevated N deposition (Berg &
Matzner, 1997; Janssens et al., 2010; Ramirez et al., 2012) was confirmed by the reduced forest
floor respiration rates close to the edge compared to the stand interior at TG and KH and by
observations of slower decomposition of needles incubated in litterbags close to the edge compared
with the inner stand at TF (Vanguelova & Pitman, 2011). However, these results conceal temporal
differences in the N-mediated response of “young” and “old” SOM turnover during the
decomposition phase (Berg & Matzner, 1997). These differences are illustrated in the 2002
respiration measurements and the second litterbag study at the N-addition site indicating higher
initial turnover of labile LF layer material and lower turnover of older H layer material under N-
additions (Figures 14-15). The mechanisms responsible for this effect are centered on the role of
soil microorganisms in SOM decomposition. The higher initial mass loss of N-enriched labile SOM
was attributed to the positive effect of N on cellulose-degrading enzymes (Carreiro et al., 2000)
reflecting the ability of soil microorganisms to utilize the easily-available carbon under higher
nutrient availability at the beginning of decay. Lower turnover of recalcitrant SOM under N
additions was attributed to increasing litter structural complexity with decay (Couteaux et al., 1998)
combined with negative effects of N on the activity of enzymes responsible for lignin degradation

51
such as phenol oxidase and peroxidase (Carreiro et al., 2000; DeForest et al., 2004) explaining
declines in microbial activity or biomass (Frey et al., 2004). However, higher respiration rates of
labile LF SOM under increasing N availability were not evident in the 2013 N-addition respiration
measurements or for samples taken from the KH and TG edges (Figures13-14). Considering that
microbial activity is strongly determined by the availability of easily metabolisable C sources
(Ramirez et al., 2012) supplied to microbes as fresh needles (Berg & Matzner, 1997) but also as
fine-root exudates (Paterson, 2003), I hypothesize that decreased input of this C source to the LF
layer, at least under N-saturated conditions, restricted the respiration rate of this layer during 2013.
This assumption relies on the observed reductions in above- and belowground C inputs at the TG,
KH and SO edges and at the N-addition site after 2002.
The suggestion that N deposition control over SOM decomposition occurs through biotic
processes, i.e. through changes in the structure and composition of the microbial community and the
functioning of decomposing enzymes, is supported by the study of Kjøller et al. (2012) at the TG
edge and by an additional study carried out in the KH edge that revealed a stronger dominance of
bacterial species over fungal species close to the edge compared with the interior concomitantly
with a decrease in forest floor respiration (D'imperio, 2011).
Podzol soils such as those found in VL, KH and SO are known to accumulate C in deep soil
layers due to leaching of dissolved organic carbon (DOC) and its interaction with iron and
aluminum oxides. This process was found to be of high importance for estimating long-term SOC
sequestration in these types of soils (Six et al., 2002; von Lutzow et al., 2006). Chronic
experimental N addition was reported to result in significant increase in DOC concentrations in
deep soil layers (Pregitzer et al., 2004; Rappe-George et al., 2013). For example, Rappe-George et
al. (2013) reported DOC concentrations in the B horizon of a Norway spruce forest in Sweden to
double from 1.5 to 3.4 mg DOC l-1
(or maximum 1 g C m-2
year-1
considering a net annual rainfall
of about 300 mm) following two decades of N additions. However, this C output flux by DOC was
ignored in this study since it is considered to be of minor importance for soil C sequestration
compared with the lowest litterfall C input of about 150 g m-2
year-1
observed in the edges.
What is the net effect of elevated N deposition on SOC stocks?
Soil organic C stocks at the edges and at the N-addition site were not consistently increasing with
increasing N deposition or continuous N additions. Neither was there a positive effect of elevated N
deposition on litterfall though SOM decomposition seemed to be reduced. The general hypothesis
that elevated long-term N deposition would increase SOC stock through increased litterfall and

52
reduced decomposition was thus rejected. These results contradict previous reports of positive
effects of long-term N additions on SOC stocks owing to increased growth and reduced SOM
decomposition (Janssens et al., 2010; Lovett et al., 2013; Pregitzer et al., 2008; Whittinghill et al.,
2012; Zak et al., 2008). However, our results are in line with those based on a meta-analysis
reporting no significant overall response of forest floor C stocks to N deposition (Nave et al., 2009),
probably because any C gains through reductions in SOM decomposition rates in our sites were
compensated by simultaneous reductions in above- and belowground C inputs.
At the intermediate-N sites (KH and SO) forest floor SOC stocks were significantly higher close
to the edges (Figure 17 and Table 2) though for SO this was likely not an effect of N deposition
alone but related to an unknown site factor (Paper I). Moreover, a decade of N additions at
Klosterheden did not change the total fine LFH C stocks though some positive effect of N additions
on C stocks of the H layer was evident (Table 4). Therefore, forest floor and mineral soil C stocks
were either unchanged or slightly increased in response to elevated N deposition at sites
characterized by intermediate-N status (Figure 19a). Since litterfall was generally unaffected by
intermediate-N deposition (Figure 11), any increase in SOC is likely a result of reduced SOM
decomposition (Figures 13-15) overcompensating reduced C inputs from roots (Figure 12).
Figure 19: A schematic diagram illustrating the effect of increasing N deposition on SOC stocks (light grey
area) and on C sequestration by selected fluxes (dark grey area) at sites characterized by (a) intermediate and
(b) high long-term N deposition loads. On the y-axis + or – indicate whether the effect on C sequestration is
positive or negative.

53
At the N-saturated sites, forest floor and mineral soil C stocks were either unaffected by N (TF) or
seemed to be reduced with increasing N input (VL and TG, Figure 17). In addition, some negative
effect of N additions on SOC of the fine LFH layer was evident in the N-addition site in 2013 (due
to less accumulation in the H layer of the N-plot, Table 4). Therefore, forest floor and mineral soil
C stocks were either unchanged or slightly decreased in response to elevated N deposition at sites
characterized by N saturation (Figure 19b) probably since C gains by reduced SOM decomposition
were equivalent to or outweighed by C losses by litterfall and roots.
The observed responses of SOC stocks to intermediate and saturated N status (Figure 19) may
be linked to the hypothesis presented in Figure 1b with the response of intermediate-N and N-
saturated sites placed to the left and to the right, respectively, of the maximum SOM sequestration
rate.
The results from papers I and II contradict those from a recent synthesis of data from N
deposition and N addition studies (Janssens et al., 2010) attributing an average 10% increase in
SOC stocks to negative effects of N addition on SOM decomposition. As shown by Hasselquist et
al. (2012), the responses of soil C processes to low and high N fertilization loads were non-linear
and complex, which questions if N fertilization studies can simulate the effect of chronic elevated N
deposition on SOC stocks. The similarities in the response of forest SOC fluxes and stocks to N
addition reported in papers I and II and synthesized here (Figure 19) demonstrate that N addition
studies can simulate long-term N deposition provided that the N-treatment lasts sufficiently long
and in small continuous doses. Long (e.g. > 15 years) N addition studies can better account for a
possible lag time in the response of certain C fluxes to N deposition and better predict whether the
net effect of N on SOC stocks is positive or negative. For example, in a 14-year N-addition
experiment reduction in total stem basal area of red spruce (Picea rubens Sarg.) became significant
only after 10 years have passed since the onset of the N applications (McNulty et al., 2005).
How does elevated N deposition affect the short-term temperature sensitivity of SOM
decomposition?
The increasing trends in the temperature sensitivities of the LF and H layers with increasing forest
floor C/N ratio at the KH edge site indicate a negative effect of increasing long-term N deposition
on the short-term temperature sensitivity of SOM decomposition.
Even though the results in Paper III do not cast additional light on the mechanisms controlling
the temperature sensitivity of SOM (Conant et al., 2011) they demonstrate for the first time that N

54
deposition acts as an important regulator of the temperature sensitivity of heterotrophic SOM
respiration. These results give further support to the inclusion of N deposition in global change
models assessing the potential positive feedback of SOM decomposition on global CO2
concentrations. However, generalizations based on the results of Paper III must be tempered by few
considerations relating to the statistical significance of the data, experimental design and the length
of the incubation period.
The relationships between Q10 and SOM C/N ratio observed at the KH edge were weak with
significant positive relationship only for the LF-layer (p=0.04) and marginally significantly positive
relationship for the H-layer (p=0.1). Including the forest sites investigated by Erhagen et al. (2013)
improved the statistical significance markedly for both layers. The steep slope of the H-layer
regression line in Figure 18 suggests that a small increase in SOM C/N ratio would have large effect
on Q10. This is a far reaching conclusion that needs further confirmation bearing in mind that only
two sites were used in the analysis and that even though 76% of the variation in the H-layer Q10 was
explained by the C/N, this relationship was mainly driven by the two data points with the highest
C/N ratios.
Higher decomposition rates for N-rich litter compared with N-poor litter (Berg & Matzner,
1997, Figures 14-15) and reduced input of labile C substrate as root exudates under elevated N
deposition (exemplified by increasing fine root biomass with distance from the edge, Figure 12)
could be indicative of decreasing forest floor labile C pools with increasing N deposition across the
KH edge. This raises the possibility that samples characterized by low C/N ratio (i.e. sampled near
the edge) had lower content of labile C prior the start of the incubation experiment and that the
observed responses in Figure 18 reflect the response of the labile SOM fraction only. We did not
measure the content of the labile LF-layer across the edge and we therefore cannot reject this
assumption completely. However, increased decomposition of labile LF-layer SOM with increasing
N deposition would probably have been reflected by increasing LF-layer thickness with distance
from the edge considering that aboveground litterfall was unaffected by distance. We measured the
thickness of the LF and H layers along one transect in the KH edge during 2013 (data not shown)
and did not observe such an increase. There was also no effect of the N-treatment on the mass of the
LF layer in the KH N-addition site by 2013. Moreover, incubation of LF and H forest floor samples
over 17 weeks during 2007 (Paper II) exhibited a linear relationship between CO2 efflux and time of
incubation indicating that the heterotrophic microbial activity was not limited by substrate

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