uef.fi
PUBLICATIONS OF
THE UNIVERSITY OF EASTERN FINLAND Dissertations in Forestry and Natural Sciences
ISBN 978-952-61-2692-0 ISSN 1798-5668
Dissertations in Forestry and Natural Sciences
DISSERTATIONS | JENIE A. GIL LUGO | MICROBIAL PROCESSES RESPONSIBLE FOR THE HIGH... | No 297
JENIE A. GIL LUGO
MICROBIAL PROCESSES RESPONSIBLE FOR THE HIGH N
2O EMISSIONS FROM SUB-ARCTIC PERMAFROST PEATLANDS AND TROPICAL SOILS AS DETERMINED BY STABLE ISOTOPE APPROACHES PUBLICATIONS OF
THE UNIVERSITY OF EASTERN FINLAND
Contrary to what was previously thought, there are habitats in the sub-Arctic tundra
emitting N
2O at exceptionally high rates.
Now, the main questions are concerning the underlying processes. In this study, stable isotope techniques were used to elucidate the
microbial pathways of N
2O production and consumption in these soils. This process data
was compared with that obtained from the largest known sources of N
2O, natural tropical
rain forest and tropical agricultural soils, in order to increase the basic knowledge on N
2O dynamics and to link the arctic ecosystem with
the global nitrogen cycle.
JENIE A. GIL LUGO
MICROBIAL PROCESSES RESPONSIBLE FOR THE HIGH N
2O EMISSIONS FROM
SUB-ARCTIC PERMAFROST PEATLANDS AND TROPICAL SOILS
AS DETERMINED BY
STABLE ISOTOPES APPROACHES
Jenie A. Gil Lugo
MICROBIAL PROCESSES RESPONSIBLE FOR THE HIGH N
2O EMISSIONS FROM
SUB-ARCTIC PERMAFROST PEATLANDS AND TROPICAL SOILS
AS DETERMINED BY
STABLE ISOTOPES APPROACHES
Publications of the University of Eastern Finland Dissertations in Forestry and Natural Sciences
No 297
University of Eastern Finland Kuopio
2017 Academic dissertation
To be presented by permission of the Faculty of Science and Forestry for public examination in the Auditorium TTA in Tietoteknia Building
at the University of Eastern Finland, Kuopio, on December 19th, 2017, at 12 o’clock noon
Grano Oy Jyväskylä, 2017
Editors: Pertti Pasanen, Matti Vornanen, Jukka Tuomela, Matti Tedre
Distribution: University of Eastern Finland / Sales of publications www.uef.fi/kirjasto
ISBN: 978-952-61-2692-0 (print) ISBN: 978-952-61-2693-7 (PDF)
ISSNL: 1798-5668 ISSN: 1798-5668 ISSN: 1798-5676 (PDF)
Author’s address: Jenie A. Gil Lugo
University of Eastern Finland
Depart. of Environmental and Biological Sciences P.O. Box 1627
70211 KUOPIO, FINLAND email: jenie.gilluo@uef.fi Supervisors: Christina Biasi, Ph.D.
University of Eastern Finland
Depart. of Environmental and Biological Sciences P.O. Box 1627
70211 KUOPIO, FINLAND email: christina.biasi@uef.fi
Professor emeritus Pertti Martikainen, Ph.D.
University of Eastern Finland
Depart. of Environmental and Biological Sciences P.O. Box 1627
70211 KUOPIO, FINLAND email: pertti.martikainen@uef.fi Professor Tibisay Pérez, Ph.D.
Instituto Venezolano de Investigaciones Científicas Centro de Ciencias Atmosféricas y Biogeoquímica Aptdo. 20632
1020-A CARACAS, VENEZUELA email: tperez@ivic.gob.ve Reviewers: Professor Nathaniel Ostrom, Ph.D
Michigan State University Depart. of Integrative Biology P.O. Box
288 LANSING, USA email: ostromn@msu.edu Professor Steven Siciliano, Ph.D University of Saskatchenwan Depart. of Soil Science P.O. Box
S7N 5A8 SASKATCHEWAN, CANADA email: steven.siciliano@usask.ca
Opponent: Reinhard Well, Ph.D
Johann Heinrich von Thünen Institute Institute of Climate-Smart Agriculture P.O. Box
38119 BRAUNSCHWEIG, GERMANY email: reinhard.well@thuenen.de
Gil Lugo, Jenie A.
Microbial processes responsible for the high N2O emissions from sub-Arctic permafrost peatlands and tropical soils as determined by stable isotope approaches
.
Kuopio: University of Eastern Finland, 2017 Publications of the University of Eastern Finland Dissertations in Forestry and Natural Sciences 2017; 297 ISBN: 978-952-61-2692-0 (print)
ISSNL: 1798-5668 ISSN: 1798-5668
ISBN: 978-952-61-2693-7 (PDF) ISSN: 1798-5676 (PDF)
ABSTRACT
Because nitrous oxide (N2O) plays an important role in the atmosphere, identifying the sources and sinks of this greenhouse gas is critical for understanding its implications across ecosystems. A better understanding of the processes that produce and consume N2O in soil is also required for improving future predictions and developing mitigationg strategies for emissions. Isotopic analysis can be used to characterize N2O emissions from soils and identify its sources including both natural abundance(δ15Nbulk; δ18O and 15N site preference - SP) and enrichment approaches (15N, 18O).
The aim of this work was to identify the microbial processes behind high N2O emissions from Sub-Arctic bare permafrost peatlands, the first known N2O source in the Arctic, using stable-isotope techniques. To achieve these goals, state-of-the art stable isotope techniques were used to elucidate the microbial pathways of N2O pro- duction and consumption in the soil. Further, I compared this process data with that obtained by stable isotopes from the largest known sources of N2O, tropical agricul- tural soils (anthropogenic) and natural tropical rain forest soils. Both natural abun- dance method (δ15Nbulk; δ18O and SP) and 15N enrichment approach were applied un- der field conditions which allowed comparison among methods while studied mi- crobial processes of N2O production and consumption in situ.
The results include the first data on the nitrogen and oxygen isotopic composition of N2O emitted from Arctic tundra. The emission-weighted average δ15Nbulk value for N2O from the Sub-Arctic bare permafrost peatlands falls within the range of the emis- sion-weighted average values from other natural ecosystems, including the Brazilian Amazon tropical forest, but is distinct from those for managed/agricultural ecosys- tems. Thus, the results were consistent with the observations that natural ecosystems emitt N2O enriched in 15N compared to fertilized soils. This implies that if in the future, a smaller rate in the overall decreasing trend of δ15NbulkN2O tropospheric
isotopic composition is found, it cannot be attributed only to agricultural N2O emission reductions from mitigation actions but also from soils in natural ecosystems that may be emitting more N2O to the atmosphere due to warmer conditions. The results also agree with the previous findings that source partitioning based on isotope bulk composition (δ15Nbulk, δ18O) of N2O emissions in situ is not straightforward mainly because the various pathways produce N2O with a wide range of isotope values.
The individual 15N site preference (SP) values from N2O were also partly outside the range of the SP values from studies using pure cultures of bacteria, but SP emision-weighted averages of N2O emitted from this study were within the range of SP values reported from soil incubation studies. Because both nitrfiers and denitrfiers are found in soils together, it is better to use the SP values from the soil incubation studies than the SP values obtained in pure culture studies to identify the processes behind N2O emissions in situ. The SP values from emitted N2O indicated a temporal shift of microbial production and consumption of N2O during the sampling period.
Soil emission SP data suggest that the N2O emission from Sub-Arctic bare permafrost peatlands are more likely to be produced by nitrifier-denitrification in the relatively drier study year with lower N2O emissions, but due to variable published SP values for N2O production processes in soils, this interpretation has to be taken with caution.
The SP values from the Venezuelan agricultal site, and from the Brazilian Amazon tropical forest fell within the SP range found for denitrification in incubation studies.
According to SP values at depth, denitrification was the main N2O production pathway in the profile of all the soils studied. The results further suggest that in per- mafrost peat profiles with high water content, a substantial fraction of N2O is reduced to N2 before reaching the atmosphere.
The results from the 15N tracer experiment in situ provided evidence that denitri- fication is mostly responsible for the high N2O surface emissions from Sub-Arctic permafrost peatlands (~79%), but also showed that production from nitrification is taking place in these soils (~20%). Nitrification is a key process involved in N2O pro- duction in these soils directly and indirectly through the production of the substrate (NO3-) for denitrification. The 15N enrichment experiment was carried out in a year with high N2O emissions. In wet years, when N2O emissions are high, denitrification is the primary source of N2O. In dry years, when N2O emissions are low, the relative contribution of nitrification/nitrifier-denitrification increases. The 15N enrichment ex- periment provided also deeper insights into N2O dynamics and general nitrogen (N) cycle processes in the permafrost peat soils. The recovery of the label in N2O in bare peat soils (BP) (maximum value ~24%) up to threefold larger than the relative portion of label observed in plants in the vegetated surrounding peat (VP) (maximum value
~ 9%), suggests that N2O production is restricted in VP by the lower availability of N associated to low N mineralization and nitrification rates and the high C/N ratio
measured in theses soils. The results suggested that the microbial demands are met in BP soils while the opposite is true for VP soils. Thus in BP soils, in the absence of plants, any excess of N is completely available for microbes and together with favor- able environmental conditions (intermediate soil moistures, low C/N, high bulk den- sity) might result in high N2O emission from these soils.
There are limitations in both natural abundance and enrichment studies and if applicable, both approaches natural abundance and enrichment, should be used to- gether to get best-possible insight into N2O biogeochemical cycles and N cycling pro- cesses in soils. In order to evaluate accurately microbial pathways producing and consuming N2O in soils, particulary to better address the usefulness of SP in partitioning microbial mechanisms in soils, further studies in soils mesocosms are required.
Universal Decimal Classification: 504.7, 546.172.5, 631.445.1, 631.445.7, 631.461 CAB Thesaurus: greenhouse gases; nitrous oxide; emission; peatlands; permafrost; arctic regions; subarctic soils; tundra; microorganisms; microbial activities; soil bacteria;
denitrification; nitrification; stable isotopes; nitrogen; tracer techniques; tropical soils;
agricultural soils
Yleinen suomalainen asiasanasto: kasvihuonekaasut; dityppioksidi; päästöt; suometsät;
ikirouta; arktinen alue; subarktinen vyöhyke; tundra; mikro-organismit; mikrobit; bakteerit;
denitrifikaatio; nitrifikaatio; isotoopit; typpi; merkkiaineet; trooppinen vyöhyke;
maatalousmaa
ACKNOWLEDGEMENTS
After an intensive period of seven months, today is the day: writing this acknowledgment is the finishing touch on my dissertation. It has been a long journey for me. From the colorful and sunshine of my beloved homeland Venezuela (10°29’N, 66°52’W) to the middle of nowhere in Finland (62°53’N, 27°40’E) and beyond, to the Russian tundra (67°03’N, 62°57’E). I must say that the journey has not been just geographical but also in the scientific arena and on a personal level. It has been a period of intense learning for me and I would like to reflect on the people who have supported and helped me so much throughout this period.
Firstly, I would like to express my sincere gratitude to my supervisors Dr.
Christina Biasi, Professor Tibisay Perez and Professor emeritus Pertti Martikainen for their continuous support, patience, motivation and immense knowledge. Their guidance helped me in all the time of research and writing of this thesis. Dr. Christina Biasi and Professor Tibisay Perez have been also a strong role model as women in science, inspiring me with their excellence, commitment and dedication to research and their academic achievements in this misogynistic world of science.
Besides my supervisors, I would like to thank Professor Nathaniel Ostrom, Professor Steven Siciliano and Dr. Reinhard Well, for enduring the task of evaluating my doctoral thesis in such a short time period and for your extraordinary support in this process.
I am grateful to all coauthors and collaborators who participated in the manuscripts included in this thesis. My special thanks to my fieldmates with whom a share endless hours of work, but also delicious meals and lot of fun.
I would like to thank fellow friends from the Biogeochemistry Research Group for their wonderful collaboration. You supported me greatly and were always willing to help me. I would particularly like to single out my good friends Maarit and Promise;
I want to thank you both for your support, encouragement and advice that helped me get through the most stressful and demanding circumstances.
I thank my fellow isotopicmates Ines Melendez, Yolmar Contreras, Miriam Diaz, Delfin Ribas, Hugo Cornejo and April Vuletich for the stimulating discussions, for the sleepless nights we were working together and for all the fun we have had. My eternal gratitude to my teacher and mentor, Hector Henriquez. He taught me more than I could ever give him credit for here. He showed me, by his example, what a good scientist and person should be.
This work would have been impossible without the financial support of the Academy of Finland, project CryoN2010-2014 (decision Nr. 132045), the European Union 7th Framework Program under project Page 21 (contract Nr. GA282700), strategic funding from University of Eastern Finland (project FiWER; Finnish- Russian Research collaboration projects), (DEFROST)-Nordic Centre of Excellence Program (Impacts of changing cryosphere – depicting ecosystem-climate feedbacks
from permafrost, snow and ice), Finnish Cultural Foundation, Vilho, Yrjö ja Kalle Väisälän Foundation and Niemi Foundation.
Last but not the least; I would like to thank my family: my parents and my sisters for supporting me spiritually throughout writing this thesis and provided me through moral and emotional support in my life. I am also grateful to my other family members and friends who have supported me along the way. Most importantly, I wish to thank my husband, Henry, for supporting my adventurous spirit and embarking with me in this journey to the other side of the world. My love and gratitude to my Santiago, my aurikopoika, who teaches me every day to embrace the differences and celebrate the uniqueness.
GRACIAS TOTALES!
Kuopio, December 2017 Jenie A.Gil Lugo
LIST OF ABBREVIATIONS
at% atom percent
BAF Brazilian amazon forest BP Bare permafrost peatland
BRICS Brazil, Russia, India China and South Africa DNRA dissimilatory nitrate reduction to ammonium
GC gas chromatography
GHG greenhouse gas
INAMEH Instituto Nacional de Meteorología Hidrología KCL potasium chloride
NO2- nitrite
NO3- nitate
NH4+ ammonium
OECD Convention on the Organisation for Economic Co-operation and Development
SP 15N site prefecences
UNEP United Nations Environmental Program WFPS water filled pore space
VSC Venezuelan savanna - cornfield VP vegetated peatland
LIST OF ORIGINAL PUBLICATIONS
This thesis is based on data presented in the following articles, referred to by the Roman Numerals I-III.
I Park S., Perez T., Boering K.A, Trumbore S.E., Gil J., Marquina S. & Tyler S.C.
(2011), Can N2O stable isotopes and isotopomers be useful tools to characterize sources and microbial pathways of N2O production and consumption in tropical soils?, Global Biogeochemical Cycles, 25, GB1001, doi:10.1029/2009GB003615.
II Gil J., Perez T., Boering K.A., Martikainen P. J. & Biasi C. (2017). Mechanisms responsible for high N2O emissions from subarctic permafrost peatlands studied via stable isotope techniques, Global Biogeochemical Cycles, 31, 172-189, 10.1002/2015gb005370.
III Gil J., Baggs E.M., Rütting T., Perez T., Marushchack M., Novakovskiy A., Trubnikova T., Kaverin D., Martikainen P.J.& Biasi C. Fate of mineral nitrogen in N2O hotspots and coldspots located on permafrost peatlands of sub-Arctic tundra: results from an in situ 15N labelling experiment. Submitted .
The above publications have been included at the end of this thesis with their copyright holders’ permission.
AUTHOR'S CONTRIBUTION
I) The author Jenie A. Gil contributed to the design of the study. She was responsible for the data collection at the Venezuelan site and also collaborated with the data processing. Sunyoung Park, Tibisay Perez and Kristie Boering wrote the first version of the manuscript after which Jenie A. Gil and the others co-authors contributed to the writing process.
II) The author Jenie A. Gil designed the study together with Christina Biasi and Tibisay Perez. She was responsible for the data collection and processing. She wrote the first version of the manuscript of the article together with Christina Biasi and Tibisay Perez after which the other co-authors contributed to the writing process.
III) The author Jenie A. Gil contributed to the design of the study. She was responsible for the data collection and processing. She wrote the first version of the manuscript together with Christina Biasi after which the other co- authors contributed to the writing process.
CONTENTS
ABSTRACT ... ERROR! BOOKMARK NOT DEFINED.
ACKNOWLEDGEMENTS ... 10
1 GENERAL INTRODUCTION ... 18
1.1 Nitrous oxides sources and sinks ... 18
1.2 Processes producing and consuming N2O in soils ... 19
1.3 Stable isotopes as a tool to characterize N2O dynamics and source partition ... 20
1.4 What do we know about nitrous oxide from arctic soils? ... 24
1.5 Relevance of N2O emissions from tropical agricultural soils ... 25
2 MATERIALS AND METHODS ... 27
2.1 Study sites ... 27
2.1.1 Sub-Arctic bare permafrost peatland – Russian tundra (BP) ... 27
2.1.2 Cornfield – Venezuelan tropical savanna (VSC) ... 28
2.2 Methods ... 30
2.2.1 Emission rates of nitrous oxide (N2O) ... 30
2.2.2 Sampling and isotopic composition of N2O emitted ... 30
2.2.3 Gas sampling from the peat profile ... 30
2.2.4 Soil nitrogen anlyses ... 31
2.2.5 Reducction of N2O to N2 and quantifyying N2O consumption in the soil profile ... 32
2.2.6 15N tracer experiement ... 33
3 AIMS OF THE STUDY ... 35
4 RESULTS & DISCUSSION ... 36
4.1 Distinguishing N2O emissions from natural and agricultural soils using stables isotopes – natural abundance method ... 36
4.2 Power of stable isotopes to characterize N2O dynamics in soils ... 39
4.2.1 N2O production processes in soils - Natural abundance approach . 39 4.2.2 N2O production processes in soils - 15N tracer approach ... 39
4.3 The key environmental factors controlling N2O emissions ... 42
4.3.1 N2O production in soils – Natural abundance approach ... 42
4.3.2 N2O consumption in soils – Natural abundance approach ... 45
4.3.3 Microbial N2O pathways from BP soils - 15N tracer approach ... 47
4.4 Why the bare surfaces of permafrost peatlands (BP) have higher N2O emission rates than the surrounding vegetated peatlands surfaces ... 49
4.5 Future perspectives for N2O emissions from the arctic with implications for
tropospheric N2O trends ...50
5 SUMMARY & CONCLUSIONS ... 52
6 BIBLIOGRAPHY ... 54
7 APPENDICES ... 63
1 GENERAL INTRODUCTION
1.1 NITROUS OXIDE SOURCES AND SINKS
Nitrous oxide (N2O) is a potent greenhouse gas (GHG) contributing to climate change. With its ozone-depleting properties (Ravishankara et al., 2009) and a global warming potential 300 times (100 year time horizon) that of carbon dioxide (CO2), N2O is the third most important GHG contributor to radiative forcing (IPCC, 2013).
Because N2O plays an important role in the atmosphere, identifying the sources and sinks of this greenhouse gas is critical for understanding its implications across ecosystems.
Atmospheric N2O concentration has risen xx% from 270 ppb in the pre-industrial era to 319 ppb, with a mean annual growth rate of 0.73 ± 0.03 ppb year-1 over the last three decades(IPCC, 2013). Globally N2O emissions are estimated to be 17.9 Tg N yr-
1, mostly from natural sources (11.0 Tg N yr-1) that account for more than 60% of the total emissions (Ciasi et al., 2013). The main natural sources of N2O are oceans(5.4 Tg y-1) and natural terrestrial ecosystems (6.6 Tg y-1). Together, natural terrestrial ecosystems, including tropical and temperate forests and grasslands/savannas, account for ~37%of the the total N2O emissions. Agricultural soils, the highest anthropogenic source of N2O to the atmosphere, contribute with ~23% (4.1 Tg N yr-
1) to the total emissions (Ciasi et al., 2013). Rivers, estuaries and coastal zones that are highly impacted by nitrogen load from terrestrial ecosystems contribute 0.6Tg N yr-
1 to the global N2O emissions (Ciasi et al., 2013). Other important anthropogenic sources of N2O include biomass burning (0.7 Tg N yr-1 ), industry (e.g., production of nylon) (0.7 Tg N yr-1) (Ciasi et al., 2013).
Nitrous oxide is stable in the troposphere and with a tropospheric lifetime of 114 years, it is transported to the stratosphere. In the stratosphere N2O is consumed by photolysis (90%) and photooxidation (10%) (Ravishankara et al., 2009). The photooxidation of N2O produces nitrogen oxides (NOx = NO + NO2) that destroy stratospheric ozone through catalytic processes (Ravishankara et al., 2009). These two photochemical reactions are the most important sink for N2O, representing ~89% of the total sink (14.3 Tg N yr-1) (Ciasi et al., 2013). Soils are also a sink for N2O (Donoso et al., 1993; Chapuis-Lardy et al., 2007; Schlesinger, 2013), accounting for around 2%
of the current estimated N2O sources (0.3 Tg N yr-1) (Schlesinger, 2013).
At present, the estimated global budget of N2O is unbalanced, showing an excess of sources over sinks. The disagreement between sources and sinks exceeds the observed increase in the N2O atmospheric concentrations and it is mainly attributed to the high spatial and temporal variability found in the N2O emissions from soils which causes inacurracies in the emission estimates. Hence, further understanding of the processes producing and consuming N2O in soil and their regulatory
environmental factors is needed, in order to better constrain the global N2O budget and to plan the mitigation strategies.
1.2 PROCESSES PRODUCING AND CONSUMING N
2O IN SOILS
In terrestrial ecosystems, the main processes responsible for N2O production are considered to be the microbial transformations of ammonia (NH3), nitrite (NO2-) and nitrate (NO3-), through classical nitrification by chemolithotrophic bacteria and denitrification by heterotrophs (Baggs, 2011). There are also alternative processes involved in N2O production in soil such as dissimilatory nitrate reduction to ammonium (DNRA) (Rüttinget al., 2011), co-denitification (Spott et al., 2011; Selbie et al., 2015), nitrification by archaea (Stieglmeier et al., 2014), heterotrophic nitrification (Baggs &Philippot, 2010), aerobic denitrification (Baggs &Philippot, 2010), anaerobic ammonium oxidation (Davidson & Seitzinger, 2006) and abiotic pathways such as chemodenitrification (Zhu-Barker et al., 2015) and abiotic decomposition of nitrate on reactive surfaces in the presence of light (Rubasinghege et al., 2011). The overall contributions of these alternative processes to the magnitude of the N2O emissions from soils is unclear but thought to be minor (Baggs &Philippot, 2010). In this work, I focused on the main pathways of autotrophic nitrification and heterotrophic denitrification.
Autotrophic nitrification is a two-step oxidative process where NH3 is oxidized to NO2- by ammonia-oxidizing bacteria (AOB) and/or archaea (AOA), and subsequently to NO3- by nitrite-oxidizing bacteria (NOB) under aerobic conditions (Baggs & Philippot, 2010). Recently, complete oxidation of ammonia to nitrate by a single microorganism (complete ammonia oxidation; comammox) has been reported (van Kessel et al., 2015). The production of N2O during ammonia oxidation can occur through two direct pathways. First, in the first step of nitrification, in ammonia oxidation, N2O can originate from the chemical decomposition of hydroxylamine, with NO as a precursor (Wrage et al., 2005). Second, N2O can be produced during nitrifier-denitrification. Nitrifier-denitrification refers to the oxidation of ammonia to NO2- with it subsequent reduction to form NO and N2O by the same autotrophic ammonia-oxidizing organism. It differs from the coupled nitrification-denitrification process, that requires the formation of nitrate and subsequent nitrate reduction by different microorganisms (Wrage et al., 2001). Whether nitrifier-denitrification can also produce N2 is still uncertain (Baggs & Philippot, 2010).
Denitrification is the major biological process in soils that returns fixed nitrogen to the atmosphere and thereby closes the nitrogen cycle. In the denitrification process, NO3- or NO2- are stepwise reduced under anoxic conditions to N2. NO and N2O are produced as intermediary gaseous products. The ability to denitrify is widespread among bacteria, and there is also fungal denitrigication (Baggs
&Philippot, 2010). Denitrification may also act as a sink for atmospheric N2O, as N2O can be reduced to N2 in complete denitrification(Chapuis-Lardy et al., 2007).
Under most soil conditions, nitrification and denitrification occur simultaneously in neighboring aerobic and anaerobic microhabitats. Then the net N2O flux to the atmosphere is the result of both processes (Conrad, 1996). More recent studies have suggested that the nitrifier-denitification process may also contribute considerably to N2O fluxes from soils under conditions of high N availability but low organic C, oxygen availability and pH (Wrage et al., 2001; Wrage et al., 2004). Whether denitrification or nitrification processes dominates depends on many factors, including soil type and vegetation cover (Stehfest & Bouwman, 2006), substrate availability (mineral N and organic C) (Wrage et al., 2004; Baggs & Philippot, 2010 ), O2 availability (Bollmann & Conrad, 1998), pH (Baggs et al., 2010), soils porosity and plant residues (Kravchenko et al., 2017) among others. Soil water content is a key control of both processes because it regulates oxygen availability in soil. Nitrification is the preferential source of N2O fluxes from well-aerated soils (WFPS < 60%), while N2O production in wet soils (WFPS 60 - 90%) is predominantly derived from anaerobic denitrification (Bateman & Baggs, 2005). Often many soils have their optimum for N2O emissions under wetter conditions than 80% WFPS (Batterbach- Bahl et al., 2013), although this depends on diffusivity related to soil texture (Davidson et al., 2000). Temperature is another important driver of N2O emissions affecting soil processes directly (e.g. effects on enzymatic processes involved in N2O production) and also indirectly (e.g. increase in soil anaerobiosis cause by temperature-induced increase in soil respiration) (Batterbach-Bahl et al., 2013).
Seasonal and spatial dynamics of soil moisture, temperature and plant activity have a significant influence on N2O emission rates. There are peaks in the N2O emissions, caused by “the transition effect” during e.g. freeze/thaw cycles and changes in the water table, with significant contributions to the annual N2O budget (Groffman et al., 2009). Nitrogen uptake by plants N affects N2O emission rates as well. In N-rich soils there is sufficient amounts of NO3- and NH4+ for microbes , and they can compete well for N with plants. In the absence of plant activity, N in soil is completely available for microbial processes (e.g. nitrification and denitrification) favouring the N2O production (Schimel et al., 2004).
1.3 STABLE ISOTOPES AS A TOOL TO CHARACTERIZE N
2O DYNAMICS AND SOURCE PARTITION
In order to better constrain the global N2O budget and to develop management strategies to mitigate N2O emissions from soils, further understanding of the N2O producing and consuming processes in soil is required. To date, distinctions between N2O production processes have relied on inhibition methods or stable isotope techniques. Several inhibitors such as nitrapyrin, chlorate and acetylene (C2H2) have been used in the past to distinguish between nitrifier and denitrifier N2O production in soils (De Boer and Kowalchuk, 2001). However, the limitations of inhibition
methods are widely known. These limitations include problems with sorption, volatilization, diffusion and rapid degradation in soil, additional carbon sources, reducction to toxic chemical species at low pH, underestatimation of denitrification due to short supply of NO3-, resulting from the inhibition of nitrification, and catalytic oxidation of NO to NO2- among others (Groffman et al., 2006; Batterbach- Bahl et al., 2013).
The limitations of the inhibitors teqniques have led to the development and adoption of alternative approaches like stable isotope techniques to characterize N2O emitted from soils and identify its sources (Perez et al., 2000; Tilsner et al.,2003; Van Groenigen et al., 2005). Stable isotope techniques include both natural abundance (δ15Nbulk; δ18O and 15N site preference (SP) ) and enrichment approaches (15N, 18O).
Source partitioning of N2O in soils using isotopic analysis is based on the isotopic fractionation of the N2O during production and consumption in soils. Biological processes, favor of 14N and 16O over 15N and 18O, respectively, causing fractionation between the light and heavy isotopes. Fractionation during production of N2O by nitrification is greater in magnitude than that associated with denitrification, meaning that N2O produced in nitrification is more depleted in 15N and 18O than N2O produced via denitrification (Baggs, 2008 and references therein). The natural abundance approach to source partitioning N2O production, especially δ15Nbulk (bulk refers to average δ15N value of the α and β N atoms; see clarification below) and δ18O, have been widely used to distinguish between different production pathways, as well as to determine the production and consumption rates of this gas in soil (Perez et al., 2006; Xiong et al., 2009). Nonetheless, distinguishing which microbial process is the predominant contributor to the N2O emissions from a particular soil based on traditional stable isotope analysis (i.e., δ15Nbulk and δ18O measurements) remains difficult because various processes may occur simultaneously in different soil microsites. In addtion, the net isotope effects associated with N2O production from nitrification and denitrification are not constant (Vieten et al., 2007; Ostrom &
Ostrom, 2012). Therefore, source attributions using traditional stable isotope analysis are inconclusive and only indicate whether a process is occurring or not.
Recent advances in stable isotope techniques have increased the precision of current natural abundance methods through the measurements of the intramolecular
“site preference" (SP) of the N2O (Yoshida and Toyoda, 2000; Bol et al., 2003; Sutka et al., 2006; Baggs, 2008). The intramolecular site preference (SP), also known as the "site specific" 15N isotopic composition of N2O or simply “15N site preference”, is defined as the difference in nitrogen isotopic composition in the two different nitrogen atoms in the N2O molecule. Nitrous oxide is a linear molecule (NNO) and 15N can be present at the central N atom position (the α site) or the terminal N atom position (the β site), and the “15N site preference” nitrogen isotopic composition is defined as the difference in δ15N of the α (δ15Nα) and β (δ15Nβ) sites. The values for δ15Nα and δ15Nβ for a given sample of N2O are determined by the N2O production mechanism (Yoshida and Toyoda, 2000; Perez et al., 2001; Sutka et al., 2004; Baggs, 2008);
therefore, it is expected that N2O production processes have distinctive SP values.
Evidence that has been provided by SP values obtained from pure culture bateria studies. There the SP values for denitrificaton range from -11 to 0‰ (Well et al., 2006;
Koster et al., 2013; Decock an& Six, 2013) while the values for nitrificatio vary from 27 to 36‰ (Sutka et al., 2006; Decock & Six, 2013; Koster et al., 2013). However, discrepancies have been found in SP values between pure culture bacteria and soil studies. For instance, SP values from soil incubation studies for nitrification range from 19‰ to 36‰ (Well et al., 2008), for nitrification/nitrifier-denitrification from - 25‰ to -8‰ (Perez et al., 2006; corrected values), and for denitrification from 1‰ to 21‰ (Well et al., 2006; Well & Flessa 2009, Perez et al., 2006; corrected values).
Although there is still some controversy regarding the difference in SP values of N2O from microbial pure culture and soil incubation studies (Oddyke et al., 2009;
Decock & Six, 2013), SP measurements have significantly increased the possibility of partitioning the processes involved in N2O production in soils. Until now, it has been assumed that SP values are neither affected by isotope fractionation nor by the 15N composition of the nitrogenous substrates (Yoshida and Toyoda, 2000; Baggs, 2008).
However, this has recently been challenged by Yang et al., (2014) based on the study of isotopic fractionation by a fungal nitric oxide reductase during the production of N2O from NO. There the SP values showed isotopic enrichment during the course of the reaction. This implies that SP values of N2O production via NO- N2O reduction step from pure culture studies can have an additional isotopomer fractionation that could cause bias in the results.
In general, the main limitations using the 15N-natural abundance (δ15N and SP) approaches are related to the overlapping isotope values of the contributing sources and incomplete information on fractionation associated with N2O production and consumption. These limitations are especially true of in situ conditions. There the temporal and spatial variation (e.g. in soil properties, microbial community composition and processes rates, soil) is high (Decock & Six, 2013; Toyoda et al., 2015).
To overcome the shortcomings of the natural abundance approach during isotopic analysis in environmental studies, additional isotopic enrichment-level approaches (15N, 18O) can be used. Enrichment approaches have been developed with the aim to quantify the contribution of the individual sources to N2O emissions. This approach focuses on distinguishing between nitrification and denitrification following the addition of a 15N tracer to the soil (Bagss, 2008). The 15N tracers are applied as single and double 15N- labeled ammonium nitrate (NH4NO3), which enables quantification of N2O emissions produced by denitrification and nitrification. Denitification is quantify from treatment with 14NH415NO3 and nitrification from the difference in 15N2O between the 15NH415NO3 and 14NH415NO3 treatments. To account for nitrification-coupled denitrification, 15NH414NO3 treatment may also be added. (Baggs et.al, 2003; Baggs & Blum, 2004; Wrage et al., 2005, Baggs, 2008). However, with the single and double 15N- labeled ammonium nitrate approach, distinguishing N2O producction from nitrification and nitifrier
denitrification remains problematic. To enable differentiation between nitrification and nitifrier-denitrification , the application of 18O-enrichment water has been proposed by Wrage et al., (2005) based on the theory of different oxigen sources (O) to nitrate (NO3-) and subsequently N2O during different stages of nitrification (Wrage et al., 2005 and references therein). However this approach should be adopted with caution due to a possible exchange of the applied 18O (enriched water) with soil water and nitrate, leading to incorrect conclusions about the origin of the N2O (Kool et al.
2007; Kool et al., 2009; Kool et al., 2011).
In addition to N2O source partitioning, it is possible to follow the flow of 15N through the different compartments of an ecosystem using the 15N enrichment approach including soil, plants and the atmosphere (gases emitted). This provides valuable information on nitrogen processes, turnover and pathways (Gardner et al., 2009; Harrison et al., 2012; Wild et al., 2015).
Whilst the 15N-enrichment approach may be desirable, since isotopic fractionation can be ignored, and it enables quantification of microbial sources of N2O without reliance on inhibitors, it also has limitations. With the 15N addition there is an exogenous N supply that could cause undesirable effects. There may also be uncertainties about uniform distribution of the label added in soil and the method is excessively expensive to be used in large scale experiments (Groffman et al., 2006).
N2O emission at the soil surface is the result of production and consumption microbial processes. In soils, N2O consumption occurs mainly through N2O reduction to N2 during the last step of microbial denitrification (Baggs & Philippot, 2010). One of the major problems to quantify N2 derived from soil denitrification comes from the large background atmospheric N2 concentrations that limits our ability to perform direct measurements of soil emitted N2 (Bouwman et al., 2013).
There are methods used to overcome this problem in laboratory studies, including acetylene-based methods, helium incubation method and 15N enrichment approach, but due to technical limitations, only the 15N enrichment approach can be used “in situ”. More recently, N2O isotopic fractionation method has been suggested for quantification of N2O consumption in soils under field conditions (Lewicka-Szczebak et al., 2017 and reference therein). This method offers the advantage that it is non- invasive and has lower cost compared to the 15N enrichment approach. The N2O isotopic fractionation method is a natural abundance apporach (δ15Nbulk; δ18O and SP) based on changes in the isotopic composition of N2O during its consumption (reduction to N2) in soil. The isotopic signatures (δ15Nbulk; δ18O and SP) change during the N2O reduction and the magnitude of the reduction is reflected in the isotopic compososition the N2O residual fraction. Thus the fraction of the N2O remaining can be calculated from the isotopic enrichment of the residual N2O, as long as the the isotopic signature of the initially produced N2O before reduction (δ0) and the enrichment factors associated with N2O reduction (ε) (Toyoda et al., 2011; Zou et al., 2014) are known. In this work, I attempt to use the N2O isotopic fractionation method
to quantify the N2O comsumption in the soil profile of natural (Arctic) and agricultural soils (Tropical) before escaping to the atmosphere.
1.4 WHAT DO WE KNOW ABOUT NITROUS OXIDE FROM ARCTIC SOILS?
The Arctic and sub-Arctic regions store more than 50% of the earth’s soil carbon pool (1300–1600 Pg C) (Schuur et al., 2015). The possible increase in release of greenhouse gases CO2 and CH4 from these carbon stocks to the atmosphere in a changing climate has been intensively studied (Schuur et al., 2009; Schuur et al., 2015; Schädel et al., 2016). However, the Arctic stores not only huge amounts of carbon but has also large nitrogen (N) reservoirs (conservative estimate: 67 billion tons ~ 60 Pg N) (Harden et al., 2012). Nevertheless, little is known about N2O emissions from cold-climate terrestrial ecosystems, such as Arctic and sub-Arctic soils.
It has been suggested that the N2O emissions from Arctic ecosystems should be negligible because of the shortage of mineral N in these soils (Ma et al., 2007; Takakai et al., 2008; Siciliano et al., 2009; Goldberg et al., 2010), the slow mineralization of organic matter in cold climates (Nadelhoffer et al., 1991) and because N deposition rates are low (Dentener, 2006). However, recent findings show that Arctic soils might also be a relevant source of N2O. Around a decade ago, hotspots of N2O emissions in sub-Arctic tundra was reported for the first time (Repo et al., 2009; Marushchak et al., 2011). These hotspots are patches of bare peat surfaces, also called “peat circles”, on elevated permafrost peatlandsand and likely developed through frost action and wind erosion without any contribution from human related activity (Repo et al., 2009;
Marushchak et al., 2011). At present, the N2O emissions from the Sub-Arctic bare permaforst peatlands range from 0.5 up to 30 mg N2O m-2d-1,with the highest rates measured during hot and moist summers (Repo et al., 2009; Marushchak et al., 2011;
Voigt et al., 2017a). The N2O emissions from these bare peat surfaces are comparable to those from drained boreal peatlands used for agriculture (0.1 – 15.1 mg N2O m2d-
1) (Maljanen et al., 2010), and to tropical forests (0.09 – 2.5 mg N2O m-2 d-1), the latter have the highest N2O emissions reported from natural ecosystems (Werner et al.,2007). The large organic N reservoirs of permafrost peatlands, the oxic conditions in the upper part of peat profile, as a result of the uplifting by the permafrost, and the favorable moisture content create optimum conditions for the N2O production in theses soils. Moreover, the absence of vegetation maybe a key factor allowing the high N2O emissions from these bare surfaces, since there is no competition for inorganic N between microbes and plants.
Denitrification has been suggested as the key process for the N2O emissions from the bare peat surfaces under favorable wet conditions (≥ 60% WFPS), (Repo et al., 2009). This was supported by results from a laboratory incubation study where addition of nitrate stimulated N2O production under anoxic conditions (Palmer et al., 2011). On the other hand, nitrification also occurs in these soils (Pitkämäki, 2010) and
enables elevated NO3- availability through the sampling season in bare peat (Marushchak et al., 2011; Voigt et al., 2017a). Nitrification in bare peat is supported by higher ammonium availability there compared to the surrounding vegetated soil (Marushchack et al., 2011).Thus, these results suggest that a combination of processes, nitification and denitrification, are responsible for the N2O production from bare peat surfaces on permafrost peatlands in the sub-Arctic region. However, both the relative contribution of denitrification and nitrification to N2O emission and the environmental and physical factors regulating the contibuation of theses proceseses remain unknown
Under global warming scenarios, it is evident that warming of the Arctic will increase soil temperatures and result in permafrost thaw. Thus, with the expected higher microbial activity derived from enhanced surface temperature, N2O production may also increase (Field et al., 2007; Schuur et al., 2009; Elberling et al., 2010, Hollense et al. 2011). There likely will be an increase in the N2O emission from the permafrost peatlands in the sub-Arctic regions (Voigt et al., 2017a). A warming experiment on the bare peat soils in permafrost peatlands showed that a small increase in growing season temperature (~1°C), without causing permafrost thaw, increased N2O emissions from previous known N2O hotspots (bare peatlands). The warming also triggered N2O emissions from the vegetated surfaces of the permafrost peatlands that do not emit N2O under present climate (Voigt et al., 2017a). In addition, the results of recent mesocosm and laboratory incubation studies show that arctic soils have a potential for high N2O emissions after permafrost thawing (~ 3 - 4 mg N2O. m-2 d-1) (Elberling et al., 2010; Voigt et al., 2017b). An estimated ninefold increase in N2O emissions from the bare peat surfaces and the surrounding vegetated peatlands can occur in warmer climate resulted from warmer soils and deeper seasonal thaw (Voigt et al., 2017b). Furthermore, high N2O concentrations in upland tundra soils facing permafrost thaw due to thermokarst formation have been reported (Abbott & Jones, 2015).
Altogether, these findings place N2O high on the agenda in Arctic research and demonstrate the need to include N2O in future Arctic GHG budgets. Now the imminent questions are related to the underlying processes responsible for the N2O emissions from Arctic soils and the global implications of Arctic N2O emissions.
1.5 RELEVANCE OF N
2O EMISSIONS FROM TROPICAL AGRICULTURAL SOILS
Nitrogen cycle has been transformed significantly in the past 50 years (Austin et al., 2013), and land use change to agricultural uses is expanding rapidly. This is particulary noticeable in tropical regions where the largest natural N2O source to the atmosphere (tropical rain forest) is located (Bustamante et al., 2014). Fertilizer use in BRICS and non-OECD countries has doubled after 1975 (Steffen et al., 2015). In Latin
America and the Caribbean (LAC) alone 8% of the world’s GHG emissions for the year 2005 were emitted (UNEP, 2010). Also, land use changes and forestry and agriculture in LAC alone represent the largest anthropogenic GHG emissions (67 % of the relative contribution from all sources). These large changes in N cycle implies that LAC countries have the potential of enhanced N2O soil emissions (Austin et al., 2013).
Trace gases emitted from tropical and temperate agroecosystems are affected not only by soil type, soil water content and temperature described in section 1.2 but also by the complexity of interactions among land management, N-fertilizer application, residue management and crop type. Measurements of trace gas emissions from tropical agrosystems, particularly those in areas where agriculture is expanding (such as LAC) are required to: 1) identify the microbial pathways of N production to select the appropriate mitigation strategies, and 2) to improve global estimates of trace gas emissions derived from intensive agriculture from these sites with emerging N2O sources.
Natural abundance nitrogen stable isotopes in N2O have been used as a tool for differentiating not only microbial processes in soils (as mentioned in section 1.3), but also as a way of differentiating natural from antrhopogenic N2O soil sources (Perez et al 2000, 2001). The atmospheric N2O burden is a result of agricultural activities that produce isotopically light N2O in comparison to tropical rain forest soils, the largest natural N2O source to the atmosphere. Unfortunately, very few N2O natural abundance stable isotope data are available from tropical agricultural soils, information that will fill the gap to better constrain the global N2O budget.
2 MATERIALS AND METHODS
2.1 STUDY SITES
2.1.1 Sub-Arctic bare permafrost peatland– Russian tundra (BP)
The study site is located in Northwestern Russia, near the settlement of Seida and 70 km southwest of the city of Vorkuta, in the Komi Republic (67⁰03'N, 62⁰57'E). It is in the discontinuous permafrost zone of the sub-Arctic East European tundra. Mean annual precipitation and air temperature are 505 mm and -5.8 °C, respectively (30 years average, data from weather stationat Vorkuta). The growing season lasts approximately 3 months, from mid-late June to early-mid September. The landscape in the area consistsof many ecosystem types and land cover classes, including upland tundra, water-logged fens and bogs, peat plateau complexes and patches of forest.
The experimental site was established on the extensive peat plateau complexes underlain by extensive discontinuous permaforst (70%–90% coverage), which characterizes the area. The site has peat deposits with a mean thickness of 2.6 m (max.
up to >4 m) (Hugelius et. al., 2012). In the peat plateaus the peatland surface has been uplifted above the ground water level by frost action (high ground ice content) and the peatland above the permaforst is mostly well drained and covered by bog vegetatation (Ronkainen et al., 2015).
The bare peat surfaces (BP) are N2O hots spots and, as such, are the focus of this study. The bare peat surfaces are patterned ground features located on the peat plateaus. They are round in shape (hence they are also called “peat circles”), with sizes ranging from 10 to 500 m2 and have only sporadic bryophytes and lichens at the surface and completely lack vascular plants. They have likely developed through frost action and wind erosion without any contribution from human intervention (Repo et al., 2009; Marushchak et al., 2011). The soil properties of the bare peat are in strong contrast to surrounding vegetated areas on the peat plateu. High soil microbial respiration (SMR) rates haven reported from these soils despite of its old age (3500-year-old C) (Biasi et al., 2014). In total, bare peat surfaces occupy about 1%
of the peat plateau area in the Seida region, where the peat plateau covers around 20% (Figure 1) (Ronkainen et al., 2015).
The study was conducted during the growing season in 2010 and 2011, N2O and CO2,surface emissions and concetrations in the peat profile, were measured from bare peat (BP) and vegetated surrounding areas (VP) once a week during the sampling period (45 days in 2010 and 57days in 2011). For stable isotope analysis, N2O samples from surface emissions and concentrations in the peat profile for natural abundance determination (δ15Nbulk, δ18O and SP) were collected every other week in 2011. 15N tracer experiment was implemented in 2010.
(a)
(b) (c)
Figure 1.(a)Bare peat surfaces on the peat plateau in the discontinuous permafrost zone of the sub-Arctic East European tundra. Close-up of one frame for static chamber measurements on (b) bare peat surfaces (BP) and (c) the vegetated peat (VP) on the surroundings.
2.1.2 Cornfield - Venezuelan tropical savanna (VSC)
The Venezuelan cornfield site was located in the Venezuelan savanna region at Fundo Tierra Nueva farm in Guárico State (9°23’33’’N, 66°38’30W). The soil in the site is classified as a Vertisol (Typic Haplusterts) and is characterized by high clay content.The area has two distinctive precipitation seasons, wet (May–October) and
dry (November–April) with ninety percent of the precipitation occuring during the rainy season and mean annual value of 1080 ± 450 mm. Mean air temperature from the region is 28 ± 7 °C (30 years average; data from INAMEH, 2013). Because the crop is rain fed, planting is scheduled during the months of highest precipitation in June–
July.
The study soil was under a no-tillage agricultural practice at the time of sampling (2005) and received the same land management for at least the previous seven years.
The cornfield site was planted and fertilized according to the typical regional farmer’s land management. Soils were fertilized with 54 KgN. ha 1 (N:P:K = 12:24:12, nitrogen as NH4+) and planted simultaneously by a planting machine with a furrow opener. In the furrow, the fertilizer and seeds are incorporated at depths from 0 to 10 cm. Thirty days after the first fertilization, soils were fertilized by broadcast addition of 18 KgN. ha-1 (as NH4NO3). At the cornfield site, gas and soils samples from surface and soil profile were collected during the period of maize growth, between May 31 and July 8, 2005. N2O fluxes were measured daily during the sampling period (35 days) using a closed chamber technique from the first fertilization until five days after the second fertilization. Specific measurements were made before and immediately after fertilization to estimate the N losses via nitrogenous gas emissions and and leaching. For stable isotope analysis, gas samples were collected once a day and soil samples once a week. In the soil profile, gas and soil samples were taken once a week (Figure 2).
(a) (b)
Figure 2. (a) Venezuelan cornfield (VSC) one month after the seeding and first fertilization. (b) Detail of set up for N2O sample collection in the soil profile.
2.2 METHODS
The methods used in this study are listed in Table 1. These were designed to study all aspects of the processes involved in N2O emissions. A short overview of techniques applied and measuasurements conducted are given below, the details are shown in the articles included in the apendix.
2.2.1 Emission rates of nitrous oxide (N2O)
N2O emission rates were determined at defined time points using the static chamber technique (Heikkinen et al, 2002). N2O fluxes were calculated based on the linear change in N2O concentration during the 40 min of chamber closure and reported as area-based flux rate. Several soil parameters were measured simultaneously, including soil temperature, soil moisture, depths of water table and active layer. An ambient gas sample was also taken associated to the chamber measurements, at about 2 m above the soil surface to determine concetration and isotopic composition of atmospheric N2O. The concentration of N2O was measured by gas chromatography (GC ) System, equipped with a (63Ni)-electron capture detector.
2.2.2 Sampling and isotopic composition of N2O emitted
The sampling for the isotope analysis was performed at the same time as the sampling for the flux measurements. Gas samples for the isotope analysis were collected from the chamber 60 minutes after enclosure (surface) and at defined depth (soil profile) using evacuated 500 mL stainless steel cylinders and a drierite/ascarite trap system, following the protocol in article I. All measurements of δ15Nbulk, 15N site preference (SP), and δ18O of N2O were performed at the University of California, Berkeley, using a Finnigan MAT 252 isotope ratio mass spectrometer (IRMS) operated in continuous flow mode, coupled with an online Finnigan preconcentrator and gas chromatograph (Thermo Finnigan, Bremen, Germany).
Isotope compositions in article I and II are reported using δ notation:
δX = (Rsample / Rstandard – 1) x 1000 ‰ (1)
for which R is the ratio of the heavy to the light isotope and X = 15N, 18O, 15Nbulk, 15Nα or 15Nβ and the 'standard' is atmospheric N2 for δ15N, δ15Nα and δ15Nβ, with the latter on the Toyoda and Yoshida (1999) calibration, (Croteau et al., 2010) and Vienna Standard Mean Ocean Water (VSMOW) for δ18O. The single measurement precision (1σ) on whole air samples containing ∼1.35 nmol of N2O was ± 0.2‰ for δ15Nbulk and δ18O and ± 0.8‰ for δ15Nα (Park et al., 2012).
2.2.3 Gas sampling from the peat profile
For soil profile sampling pits were dug in the study sites. The size of the pits and the sampling depth varied between the sites. In theVenezuela corn field site (VSC), we dug 1 pit of 1.9 m depth in which we inserted 2 m of 1/8 in. O.D. stainless steel tube probes at 10, 25, 50, 75 and 100 cm on one wall of the pit (see full description in article I). In the Russian tundra site, we dug 3 pits in BP and 3 pits in the vegetated peat (VP) soils and gas collectors (perforated PVC tubes, diameter 5 cm, and volume 250 cm3) were inserted horizontally into the pits walls at depths of 5, 10, 25, 35 and 45 cm and the pits were then closed. In addtion sensors were installed in one soil profile from BP and VP to measure soil temperature, soil moisture and oxygen (see full description in article II).
Gas and soil samples were collected from the soil profiles to determine the concentration and stable isotopic compositions of N2O, mineral N (NH4+ and NO3-), total carbon (TC) and total nitrogen (TN). Samples from the soil profile were collected on the same day as the gas emissions.
2.2.4 Soil nitrogen analyses
Soil samples were taken from near the flux chambers for mineral nitrogen analysis (NH4+ and NO3−; n=3) and analysis of δ15N in NH4+ and NO3−. This sampling was done simultaneously with the gas sampling in order to enable calculation of the instantaneous isotope enrichment factors for 15N in N2O (Mariotti et al., 1981).Values of δ15N for NH4+ and NO3- from the natural abundance approach and the 15N tracer experiement were determined using the microdiffusion method (Herman et al., 1995).
Briefly, for analysis of atom% 15N (at %) of NH4+, the soil extract was placed in a closed container and MgO was added to buffer the solution to yield a pH of ~10.5.
Volatilized ammonia (NH3) was trapped on acidified filter paper (Whatman 589/3, ashless) during an incubation period of eight days at 30 °C with shaking at 200 rpm.
For the measurements of at % 15N of NO3-, after the removal of NH4+ following the procedure above, Devarda's alloy was added to convert NO3- to NH4+ which was then trapped similarly to ammonia. After incubation, the filter paper was removed from the solution, dried for 24 hours over an atmosphere of concentrated H2SO4 in a des- iccator, and wrapped in a tin capsule. The filters were then analyzed at the University of Eastern Finland by an elemental analyzer coupled to an isotope ratio mass spec- trometer (EA-IRMS), which included a ThermoFinnigan DELTA XP Plus IRMS, Flash EA 1112 Series Elemental Analyzer, and a Conflow III open split interface (Ther- moFinnigan, Bremen, Germany). The 15N data were expressed as δ15N (‰) for natural abundance soil samples and at% 15N excess relative to the natural abundance 15N content of NO3- and NH4+ in the soils for the 15N labeling experiment. Dried bulk soil samples were also analyzed for total N and 15N content using the same EA-IRMS, and at% 15N excess values were calculated as described above (and see equation 2
