UNIVERSITY OF COPENH AGEN FACULTY OF SCIENCE
UNIVERSITY OF COPENH AGEN
CENTER FOR PERMAFROS T
Master thesis Miriam Antonia Charlotte Helbig
Changes in microbial activity due to thawing of permafrost from Peary Land, Greenland, under aerobic and anaerobic conditions
Academic advisors: Anders Priemé, Professor Morten Schostag, Ph.D. student
Submitted: 06/12/16
1
ABSTRACT
Permafrost-affected soils contain enormous amount of stored organic material. A rise in global temperatures leads to increased thaw of the ground and release of buried nutrients, which are then used by microorganisms for decomposition, resulting in the release of greenhouse gases to the atmosphere and the establishment of a positive feedback mechanism. Yet the knowledge about changes in microbial activity in thawing permafrost in the high Arctic remains sparse.
The objective of this thesis is to examine changes in microbial activity in permafrost from the high Arctic by incubating the soil under controlled conditions over six months under aerobic and anaerobic conditions. Activity was measured by monitoring fluxes of carbon dioxide (CO2), methane (CH4) and nitrous oxide (N2O). Soil properties were analyzed along with the microbial community using qPCR and Illumina MiSeq sequencing based on isolated DNA. These analyses were mainly done on the soil before incubation due to the limited timeframe of this thesis.
Results showed that there was no activity under anaerobic conditions, only under aerobic. The production of CO2 was highest and increased with depth. CH4 production was low, while N2O production was nearly non-existing. The soils organic content was generally low, but increased with depth as well in addition to soil moisture. This suggests that buried nutrients were released due to thawing and stimulated microbial activity. Also, CO2 production was significant correlated to the soils content of organic nitrogen and phosphorous content, ammonium concentration, soil moisture and conductivity.
Molecular analyses showed greater diversity of bacteria than archaea with increasing abundance with depth. The dominant bacterial phylum was Proteobacteria, followed by Actinobacteria. Methanogenic archaea were nearly absent which explains the low production of CH4. No fungi were present.
Taken all together, these results show that even in the nutrient poor high Arctic, permafrost thaw, due to increased temperatures, will result in the release of buried nutrients thereby stimulating microbial decomposition and increase the production of greenhouse gases. It is important to note that this thesis only focuses on a single soil core and does not reflect any comprehensive survey of permafrost environments.
2 RESUMÉ
Jord påvirket af permafrost indeholder store mængder af ophåbet organisk materiale. En stigning i globale temperaturer vil medføre øget optøning af jorden og frigive begravede næringsstoffer som så kan bruges af mikroorganismer til nedbrydning, som resulterer i frigørelsen af drivhus gasser til atmosfæren og etableringen af en positiv feedback mekanisme. Dog er viden omkring ændringerne i mikrobiel aktivitet i optøende permafrost i høj Arktis sparsomt.
Formålet med dette speciale var at undersøge ændringer i mikrobiel aktivitet i permafrost fra høj Arktis ved at inkubere jord under kontrollerede forhold i seks måneder under aerobe og anaerobe forhold. Aktivitet blev mål ved overvågning af forandringer af karbon dioxid (CO2), metan (CH4) og lattergas (N2O). Jordegenskaber blev analyseret sammen med det mikrobielle samfund ved brug af qPCR og Illumina MiSeq sekventering baserende på isoleret DNA. Disse analyser blev hovedsageligt gjort på jord før inkubationen på grund af den begrænsede tidsramme af specialet.
Resultaterne viste ingen aktivitet under anaerobe forhold, kun under aerobe. Produktionen af CO2 var den højeste og stigende med dybden. CH4 produktion var lav, mens N2O produktionen blev næsten ikke produceret. Jordens organiske indhold var meget lav, men steg med dybden sammen med vand indholdet. Dette tyder på at begravede næringsstoffer blev frigjort på grund af optøning og stimulerede mikrobiel aktivitet. Desuden var CO2 produktionen signifikant korreleret til jordens indhold af total organisk nitrogen og fosfat, ammonium koncentration, vand indhold og konduktivitet.
Molekylære analyser viste en større diversitet af bakterier end archaea med stigende overflod med dybden. Det dominerende bakterielle phylum var Proteobacteria, efterfulgt af Actinobacteria.
Metanogene archaea blev næsten ikke fundet som kan forklare den lave produktion af CH4. Svampe var ikke til stede.
Taget alt sammen viser disse resultater at selv i den næringsstoffattige høj Arktis vil permafrost tø, på grund af øgede temperaturer, medføre frigørelse af fangede næringsstoffer dermed stimulere mikrobiel nedbrydning og øge produktionen af drivhus gasser. Det er vigtig at notere at dette speciale kun fokuserede på en jordkerne og afspejler ikke nogen omfattende overblik af permafrost miljøer.
3 TABLE OF CONTENT
1 INTRODUCTION ...... 7
1.1 Objective ...... 8
1.1 Hypotheses ...... 8
2 BACKGROUND INDFORMATION ...... 9
2.1 High Arctic – Its climate conditions & soil properties ...... 9
2.2 Permafrost...... 10 2.2.1 A physical state & its conditions ...... 10 2.2.2 Carbon & nitrogen storage ...... 12
2.3 Microorganisms in the Arctic ...... 13 2.3.1 Function & cold-adaptation ...... 13 2.3.2 Microorganisms in permafrost ...... 15 2.3.3 Microbial activity ...... 16
2.3.3.1 Carbon dioxide (CO2)...... 16
2.3.3.2 Methane (CH4)...... 17
2.3.3.3 Nitrous oxide (N2O) ...... 17
2.3 Climate change ...... 17 2.4.1 Overall effect on the Arctic ...... 17 2.4.2 Greenhouse gases & their warming potential ...... 19 2.4.3 Global warming‟s effect on permafrost-affected soils ...... 20 2.4.4 Global warming‟s effect on microorganisms ...... 21
3 MATERIALS AND METHODS ...... 23
3.1 Field site ...... 23
3.2 Soil core ...... 24
3.3 Incubation experiments ...... 25 3.3.1 Aerobic incubation ...... 25 3.3.2 Aerobic incubation ...... 25 3.3.3 Sampling of produced gas ...... 26
4 3.4 Soil properties ...... 26 + - 3.4.1 NH4 , NO3 , DOC & pH ...... 26 3.4.2 Conductivity ...... 27 3.4.3 Soil moisture, total C, total N & total P ...... 27 3.4.4 Temperature ...... 28
3.5 Molecular analyses ...... 28 3.5.1 DNA isolatoon ...... 28 3.5.2 PCR test run ...... 28 3.5.3 qPCR ...... 29 3.5.4 Illumina MiSeq sequencing ...... 30
3.6 Data analyses ...... 31
4 RESULTS ...... 32
4.1 Soil properties ...... 32 4.1.1 Temperature ...... 32 4.1.2 Total C, total N & total P ...... 34 4.1.3 Conductivity & soil moisture ...... 35 4.1.4 pH ...... 36 4.1.5 DOC ...... 37 - 4.1.6 NO3 ...... 38 + 4.1.7 NH4 ...... 39
4.2 Incubation experiments ...... 40 4.2.1 Aerobic incubation ...... 40 4.2.2 Anaerobic incbuation ...... 40
4.3 Molecular analyses ...... 42 4.3.1 PCR test run ...... 42
4.3.2 qPCR ...... 43
4.3.3 Illumina MiSeq sequencing ...... 44
5 DISCUSSION ...... 46
5.1 Statistical erros ...... 46
5.2 Soil properties ...... 46 5.2.1 Soil temperature, moisture & conductivity indicate depth of active layer ...... 46
5.2.2 pH ...... 47
5 - + 5.2.3 DOC, NO3 & NH4 ...... 47 5.2.4 Total C, total N & total P ...... 48
5.3 Molecular analyses ...... 49
5.3.1 CO2 flux data ...... 49
5.3.2 Statistical correlation between CO2 flux data & soil properties ...... 50
5.3.3 CH4 flux data ...... 50
5.3.4 N2O flux data ...... 51
5.4 Micobial community ...... 52 5.4.1 Archaeal diversity ...... 52
5.4.2 Bacterial diversity ...... 52
5.4.3 Community composition affected by soil properties ...... 53
6 CONCLUSION ...... 55
7 PERSPECTIVES ...... 56
8 ACKOWLEDGEMENT ...... 57
9 REFERENCES ...... 58
10 APPENDIX ...... 72
6 1 INTRODUCTION
Little is known about the soils in much of the Arctic due to the very harsh climate conditions and relative inaccessibility, which make it difficult to study the environment. The Arctic in general defines a cold, harsh and windy environment, with temperatures too cold all year round to sustain trees (Crawford 2008).
Permafrost covers about 25% of the Northern Hemisphere‟s land area (Tarnocai et al. 2009) and stores approximately 50 % of the global below-ground organic carbon pool as a result of sequestration (Hollesen et al. 2011). Carbon sequestration is the process involved in the removal of carbon from the atmosphere in by capturing and long-term storage of atmospheric carbon dioxide (CO2) or methane
(CH4) (Yu et al. 2008). This promotes the interest in understanding how the arctic terrestrial carbon balance will respond to climate change.
Modeling of permafrost indicates that as much as 90 % of the near-surface Arctic permafrost could thaw by the year 2100 (Lawrence & Slater 2005). Under normal climate conditions, the ground remains cold enough to keep decomposition very slow, thereby limiting microbial growth. Warming initially accelerates decomposition, since the stored nutrients are made available for microorganisms upon thawing to be degraded and metabolized, eventually leading the soil to go from being a carbon sink to a carbon source (Sistla et al. 2013). Some of the stored organic matter is then transformed into potent greenhouse gases such as CO2, CH4 and nitrous oxide (N2O) (Zimov et al. 2006; Schuur et al. 2009; Elberling et al. 2010, Elberling et al. 2013). The fate of stored carbon will partly depend on the local environmental conditions, as the relative production of CO2 and CH4 varies according to soil water and oxygen content. Wetter soil conditions due to thaw have been implicated for causing increased production of CH4 and N2O due to less aerobic conditions (Lal 1995, Johnson et al. 2005).
The relatively inaccessibility of the Arctic leave the microbiology of permafrost remains relatively unexplored (Steven et al. 2006, Wagner 2008). The concerns about potential large scale positive feedback loops by global warming makes it crucial to gain in-depth knowledge of the microbes inhabiting permafrost environments. The challenge is to address the ecology of this unique microbial ecosystem.
The potential and actual thawing of arctic permafrost under current rates of climate change is causing considerable concern. By looking at the effect of permafrost thawing we are interested in the regional ecology, water balance and the global balance of carbon uptake and release (Van Horn et al. 2014). Microorganisms in polar regions are active and respond to change. Measurement of production and distribution of CO2, CH4 and N2O (Gregorich et al 2006, Shanhun et al. 2012) is a critical step in understanding how ecosystems contribute to and may react to climate change (Brummel & Siciliano 2011). They may change in response to altered resources with an altered bacterial community composition (Larose et al. 2013) and increased carbon and nutrient cycling occurring. It remains
7 unclear how permafrost microbial communities will respond and possibly contribute to future permafrost thaw.
1.1 Objective
This thesis focuses on the change of microbial activity in a Greenlandic permafrost soil core, extracted from Peary Land, the most northerly ice-free region on Earth. Soil microbial activity is expressed by measuring the production of CO2, CH4 and N2O. The aim of this thesis is to give an estimate on how global warming will affect high Arctic permafrost microbial communities in the longer term. This is done through incubation of permafrost soil under aerobic and anaerobic conditions at increased, but constant temperature over 6 months. During this experiment, the gas emission was measured using gas chromatography. Chemical analysis of soil properties were performed to test if they influence microbial activity. Molecular analyses of the microbial community were done as well. QPCR was performed for clone library analyses of the bacterial 16S rRNA gene and the fungal Internal Spacer Region 2 gene to giving a quantitative estimate of the microbial community, while Illumina MiSeq sequencing was used to look into its diversity, giving a qualitative estimate.
1.2 Hypotheses
First, I hypothesize that thawing of high Arctic permafrost soil will lead to the increased availability of nutrients thereby stimulating microbial activity over time. The decomposition of buried carbon and nitrogen stores will lead to the emission of CO2 under aerobic conditions and CO2 & CH4 under anaerobic conditions to the atmosphere (Gray et al. 2014). I expect at the beginning of the incubation experiment to have a high release of trapped greenhouse gases as a consequence of thawing.
Second, I hypothesize to find high microbial activity at the beginning of the incubation experiment due to the increase availability of nutrients, which declines over time as nutrients become limited.
Third, I hypothesize that due to the lack of vegetation in the high Arctic, the content of organic material in the active layer is very low.
Fourth, I hypothesize to find a higher diversity and abundance of microorganisms within the upper soil layers and decreases with depth. I expect to find a greater diversity of bacteria than archaea and fungi, mainly belonging to the phyla Proteobacteria, Acidobacteria, Actinobacteria, Chloroflexi, Bacteroidetes and Firmicutes, whereas I expect very low abundance of fungi and archaea, primarily belonging to the phylum Euryarchaetoa.
Finally, I hypothesize that microbial activity decreases with soil depth as a result of expected lower diversity in deeper soil layers as well as decreasing nutrients concentration with depth.
8 2 BACKGROUND INFORMATION
The following chapter gives an overview about the various conditions in permafrost areas that could influence the thesis objectives. It is impossible to mention all possible effects therefore I am focusing on the soil properties and conditions in the high Arctic and permafrost-affected soils as well as microbial function in and adaptation to those ecosystems. At the end, I am describing the current knowledge about climate change and its affect on the Arctic environment as well as its importance for microbial activity.
2.1 High Arctic - Its climatic conditions & soil properties
Tedrow (1970) classified soils in the polar region under four types: tundra, sub-polar desert, polar desert and cold desert, with the polar desert zone taking over at about 80 °N. The polar desert in the Arctic includes all regions north of 80 °N and is a synonymous for high Arctic (Passarge 1920).
The high Arctic is characterized by very low temperatures, low precipitation, low available moisture and negligible biological activity (Campbell et al. 1998). According to van der Wal & Hessen (2009) the high Arctic is a geologically young ecosystem with a short seasonal window for biological production. Biological activity is mainly depending on the degree of sunlight and the amount of available water, which are the greatest during the summer period. The time period for biological activity progressively decreases from about 3.5 to 1.5 months from the southern boundary of the Arctic to the north and mean July temperature decreases from 10-12 °C to 1.5 °C (Friedmann 1982).
The period over which water may be available can vary between only a few days up to several months a year, or might not be available at all, depending on the location. Mean annual air temperatures vary greatly according to location, even at the same latitude (ACIA 2004). Coastal regions are slightly warmer because of their closer proximity to open ocean areas and the frequent passage of cyclones (low-pressure winds) (Serreze & Barry 2005) and frequent experience of freeze-thaw cycles (Jónsdóttir 2005).
The soil temperature is directly related to the air temperature following a delayed increase. A change in air temperature can thus have a modifying effect on soil temperature, but others factors such as vegetation, snow cover, soil moisture and presence of permafrost affect the soil temperature as well, leading to a vertical temperature gradient within the soil (Harris et al. 2008).
Annual precipitation in the Arctic decreases from south to north and from coast to inland, yielding only 100 – 200 mm in the high Arctic (Jónsdóttir 2005), mostly in solid form as snow, which then is often blown away due to strong winds. These conditions prevent the leaching of salts and carbonates; accumulation of these can lead to salinization and calcification on the soil surface (Herbert et al.
9 2015). Regions near the coast have a higher input of salts from the marine environment compared to further inland regions. Most of the soils nitrate content is considered to be derived from the sea, but can also arrive from precipitation in the form of snow (Heikoop et al. 2015).
The surface soil in the high Arctic typically is covered with pebble sized rocks and has a low organic content (Campbell et al. 1998). Snow and ice cover on the surface can buffer the soil against the harsh conditions in the high Arctic and at the same time also act as a greenhouse as well as filtering incoming UV radiation (Cockell et al. 2002).
The annually production from any given biological process in a polar organisms is a function of the length of the growing season. During summer, the sun never goes down, whereas in winter the sun never comes up. The growing season is therefore restricted to a few months a year. Since sunlight does not penetrate below the first two cm of soil, solar energy is restricted to the surface organisms. Phototrophic bacteria and algae can thus be found on the soil surface, but they require moist surfaces, which are generally lacking in the high Arctic. In deeper soil layers, microorganisms use reduced organic compounds and inorganic materials as energy source instead; both under aerobic and anaerobic conditions (Crawford & Newcombe 2008).
2.2 Permafrost
2.2.1 A physical state & its conditions
Permafrost is a physical state of the lithosphere, characterized as ground that remains below 0 °C for at least two consecutive years and can vary in depth, reaching between 200 m and 700 m (Zhang et al. 2008). It is a thermal condition which is highly dependent on climate. The age of permafrost can be up to 1-3 million years in the Arctic and even older in Antarctica. Permafrost occurrence depends on temperature and its distribution and thickness respond to natural environmental changes and disturbances that cause alterations to the ground thermal regime (Farbrot et al. 2013).
The main importance of permafrost in the context of terrestrial biology lies in its modification of overlying habitats, in particular by preventing drainage. Permafrost underlies about 25 % of Earth‟s land surface (Jansson & Taş 2014) and is usually associated with a moister layer on top of it, called the active layer, which undergoes seasonal thawing and refreezing (French & Shur 2010) and can vary in thickness from a few centimeters to several meters (Jansson & Taş 2014). During the growth season, the active layer thaws due to increased temperatures and the ice in the ground will melt, making liquid water available for microorganisms. Water can also be available through snow melt (Dobinski 2011). The degree of thaw depends mainly on the air temperature, but is also influenced by other factors such as snow coverage, vegetation, topography and drainage (Hinkel & Nelsen 2003).
10
Figure 1: Permafrost areas in the northern hemisphere (Brown et al. 1997). Dark blue color shows the distribution of continuous permafrost (a continuous sheet of frozen material; extends under all surfaces except large water bodies), middle blue color shows the distribution of discontinuous permafrost (permafrost is broken up into separate areas surrounded by unfrozen ground) and light blue color shows the distribution is isolated permafrost patches (French 2007). The circle marks Peary Land, the area in which the soil core that had been used as material in this thesis was collected.
Most permafrost contains no liquid water but solid, frozen ice instead. Such permafrost is called dry permafrost. Since life depends on liquid water, permafrost is one of the most extreme environments on Earth. Water does not always freeze at 0 °C, but permafrost occurs at that temperature (Van Everdingen 2005). 2-7 % of water inside permafrost persists as briny liquid films, having a high content of salts which depress the waters freezing point. Water can then move through the soil by capillary flow, creating the so called wet permafrost, in which microorganisms can still be active (Ugolini & Anderson 1973, Bakermans et al. 2003). However, 95 % of the soil high Arctic soils are not moistened by liquid water (Campbell et al. 1998). In addition, permafrost is characterized by constant negative temperatures, inaccessibility of nutrient supplies, and complete darkness (Thomas et al. 2008).
The grounds thermal gradient allows water to migrate upwards during winter and downwards during summer. A portion of the ground at the top of permafrost may thaw from time to time and is called the transient layer (Shur et al. 2005). Unfrozen water moves in the direction of colder temperature by the concept of capillarity, accumulating in the transient layer. As freezing takes place, energy is released from the water, creating a negative pressure, causing the water to migrate. This effect is called cryosuction (French 2007). Permafrost acts as an impermeable layer, thereby stopping the migration.
11 Re-freezing of the ground in autumn in regions underlain by permafrost would cause the water to be trapped due to two-sided freezing: downward from the surface above and upward freezing from the perennially-frozen ground beneath. During soil freezing, cryosuction result in the formation of ice lenses resulting in water being confined progressively in a smaller space (Burn 2012). As water containing high concentrations of salts does not freeze at 0 °C due to the depression of the freezing point, the salts might be deposited in the transient layer.
2.2.2 Carbon & nitrogen storage
In the terrestrial environment, soils are the largest reservoirs of carbon. The cold temperature in polar regions slows down chemical weathering and the rate of adding organic matter. However, arctic ecosystems still tend to accumulate organic matter (mainly carbon) and other elements, due to slow decomposition and mineralization processes that are equally inhibited by the cold, dry soil environment (Jonasson et al. 2001).
Permafrost may have high organic matter content in the form of peat, root material and other frozen vegetation which was deposited over thousands of years in the Earth‟s past (Zimov et al. 2006). The low temperature in these deposits inhibits microbial mineralization of organic matter and its carbon content is considered to be “locked away” from the contemporary carbon cycle (Zimov et al. 2009).
Cryogenic soil processes such as cryoturbation (mixing of soil as a result of freeze-thaw events) will lead to the formation of permafrost affected soils of granular structure, in which organic matter will accumulate at the soils surface, but also transported down the soil profile (Kimble 2004). Permafrost affected soils store between 25 and 50 % of the global soil organic carbon pool (1.672 Pg), of which approximately 88 % (1.466 Pg) exists in perennially frozen soils (Tarnocai et al. 2009). These soils generate less biomass compared to temperate soils, which lets them function as carbon sinks through the sequestration of the organic matter. Up to now no estimation has been made of how much nitrogen (N) is stored in permafrost, only that the concentration of nutrients, especially nitrogen, phosphorus and potassium, in arctic soils are generally low (Wagner 2008; Masson-Delmotte et al. 2012).
Organic carbon derived from terrestrial vegetation must be incorporated into the soil and permafrost to be effectively stored. The carbon is stored inside the cryosphere until thawing of the ground releases it (Zimov et al. 2009). Depending on the age of the permafrost, the carbon stores inside have an equivalent age; e.g. Tedrow (1970) estimated that the organic matter in surface horizon of polar desert soil in north-west Greenland (Inglefiled Land) yielded an age of 3.300 +/- 110 years before present, whereas much of Canada was covered by ice sheets for most of last 100.000 years and permafrost only forms after deglaciation (Burn 2012).
12 Carbon sequestration is the combination of both capture and storage of carbon either as CO2 or CH4.
The only requirement for sequestration is that CO2 and CH4 are converted into some other organic chemical form. The enormous stores of organic carbon and nutrients have accumulated in the permafrost over thousands of years (Chapman 2010). Carbon and nutrient storage is the result of two primary processes:
(1) Aggregation of organic matter in form of litter as the result of aeolian or fluvial sedimentation and/or peat formation. Some of this decomposes as a result of biological activity, but a large portion of the litter builds up on the surface, forming an organic soil horizon leading to the thickening of the active layer. During autumn and cooling of the atmosphere, the ground freezes and might not thaw as much the following year (Bockheim & Tarnocai 1998).
(2) Due to the freeze-thaw cycles and cryoturbation the soil will be mixed, leading to the movement of soluble above- and belowground organic material deeper within the soil profile. Movement of soluble organic matter in water is small, but downward because of gravity along the thermal gradient, towards the freezing front (Kokelj & Burn 2005). Once the organic material has moved down to the cold, deeper soil layers, where very little or no biological decomposition takes place, it will be preserved (Tarnocai 2009).
2.3 Microorganisms in the Arctic
2.3.1 Function & cold-adaptation
Microorganisms are ubiquitous and perform a number of roles within ecosystems such as decomposition of organic material. Decomposition is the process of soil organic material (SOM) mineralization, breaking down the organic material with end products being gases as well as nutrients, + such as phosphorous and ammonium (NH4 ), required by other organisms such as plants for growth (Bardgett et al. 2005). The produced gases are released back into the atmosphere (e.g. the mineralization of carbon consequently lead to the production and release of CO2). This process is referred to as soil respiration (Mackelprang et al. 2011). After photosynthesis, soil respiration is the second largest flux of carbon and includes both root and microbial respiration (Davidson et al. 2002). The major factor controlling the mineralization of SOM is microbial activity, which is sharpened by environmental conditions. Also, the molecular structure of microbial biomass and SOM has long been thought to determine long-term decomposition rates in mineral soil. Fresh nutrient inputs will stimulate microbial activity, leading to faster decomposition of older organic matter (Schmidt et al. 2011, Wild et al. 2014).
High Arctic soils are slightly alkaline, usually with a pH above 8 throughout the soil profile. The soil pH is an important regulator of microbial activity (Heynes 1986) and the composition of microbial
13 population (Paul & Clark 1996). It has a strong effect on the structure and diversity of soil bacterial communities (Fierer & Jackson 2006, Lauber et al. 2009, Chu et al. 2010, Zinger et al. 2011). A correlation between bacterial community and soil pH can be related to carbon and moisture availability, which co-varies with soil pH (Rousk et al. 2010).
Temperature and moisture are the major determinants of the rate of decomposition since they directly influence the activity of soil microorganisms. With increased temperature the rate of decomposition will rise (Wild et al. 2014), but can simultaneously decrease under very wet conditions as soil becomes anaerobic (Shur et al. 2005, Elberling et al. 2013). Decomposition rates are limited by the availability of oxygen (Illeris et al. 2003, Conant et al. 2011) and soil moisture. When availability is low decomposition will be inhibited, resulting in accumulation of organic matter due to low primary production (Chapman 2010).
Besides temperature and moisture, the organic carbon content and degree of salinity affects the productivity of microbial biomass as well. Soil salinity has shown to act as a microbial stressor in other environments by exerting a primary limitation on water availability. It can also lead to high internal levels of ions that are toxic to metabolic activities (Zahran 1997, Yan et al. 2015).
Microbes are highly adaptable organisms capable of withstanding the harshest conditions on Earth (Lewis et al. 2007). The ability to resist freezing (and to restore activity after thawing) and the ability to metabolize below the freezing point are fundamental microbial adaptations to cold climates prevailing at high latitudes (Rivkina et al. 2000, D‟Almico et al. 2002). Cold-adapted microbial species are characterized by remarkably high resistance to freezing through adjustments in protein structure and membrane composition, production of cryoprotectants, increased variety and number of chaperones as well as up regulating expression of genes encoding cold-chock proteins (Aanderud et al. 2013, De Maayer et al. 2014). Cell viability depends dramatically on the freezing rate, which defines the formation of intracellular ice crystals (Mazur 1980, Kushner 1981).
Subsurface microbial activity is affected by soil porosity. Pores are required for movement of liquid water, which can mobilize nutrients within the soil; larger pores are associated with increased availability of organic carbon for microorganisms. This selects for oligotrophic microbial populations. Sequestration of liquid water as ice in permafrost reduces porosity and may act to limit the availability of organic carbon, but oxygen as well (Öqvist et al. 2009). Without liquid water, the majority of cellular biocatalysts such as DNA, RNA, enzymes, semi-fluidic membranes etc. remain functionally disabled (Kushner 1981).
Microbial communities in extreme environments often have lower diversity and specialized physiologies suggesting a limited resistance and resilience to change (Van Horn et al. 2014).
14 2.3.2 Microorganisms in permafrost
Morita (1975) characterized permafrost microorganisms as psychrotrophes (psychrotolerant mesophiles), meaning that they are less sensitive to low temperature than similar species from lower latitudes. More recently they have been characterized as „cryophiles‟, which are capable of growth and reproduction at low temperatures, typically ranging from -17 °C to +10 °C (Feller & Gerday 2003).
Bacteria are perhaps the most widespread and numerous of life forms on the planet and generally higher in diversity than archaea or fungi (Deming 2002). The most commonly phyla to occur in permafrost affected soils are Proteobacteria, Acidobacteria, Actinobacteria, Chloroflexi, Bacteroidetes and Firmicutes (D‟Almico et al. 2006, Steven et al. 2007, Johnson et al. 2007, Yergeau et al. 2010, Hinsa-Leasure & Bakermans 2013). The populations vary though with the depth of the permafrost.The predominance of these phyla suggests that they are equipped for cold survival (Jansson & Taş 2014). Freidmann (1994) defined psychrophilic communities as a community of survivors.
Besides bacteria, archaeal methanogens such as Methanobacterium veterum and M. arcticum have been isolated from permafrost soils, suggesting that permafrost is a favorable environment for the production of methane, a process called methanogenesis (Wagner 2008). Due to prevailing low nutrients conditions, fungi live in a dormant state as spores (small unicellular condia) (Vorobyova et al. 2001) which enable long-term survival and burst when fresh organic matter enters the ecosystem. Isolates include members of the genera Penicillium, Geomyces, Cladosporium and Aspergillus (Ozerskaya et al. 2009).
It has often been assumed that microorganisms in permafrost are in a state of anabiosis (lack of metabolic activity) (Vorobyova et al. 1997), but the physical structure of permafrost makes metabolic activity possible. Permafrost contains various geomorphological structures including cryopegs and ice wedges that harbor microbial populations (Steven et al. 2006). As mentioned previously, permafrost may contain unfrozen, briny water, which makes mass transfer of ions and nutrients possible inside the permafrost, consequently leading to nutrient uptake by microorganisms stimulating in metabolic activity (Rivkina et al. 2000). Permafrost microorganisms tend to be more halotrophic (Rivkina et al. 2004). Prince‟s (2007) detection of halophilic organisms in moderately saline permafrost provides circumstantial evidence that the primary microbial habitat in permafrost exists as thin saline liquid water veins surrounding soil particles.
Due to constant subzero temperatures, permafrost environments are an ideal system for DNA preservation. Detection of DNA sequence is not conclusive evidence that the organisms are active or alive (Rivkina et al. 2004). According to Willerslev et al. (2004), DNA concentrations and taxonomic diversity were found to decrease with age up to 400-600 thousand years. In their study, all identified bacterial taxa were known soil inhabitants, indicating that permafrost is a non-extremophile
15 environment. They concluded that PCR based methods is able to detect DNA from living bacteria, as well as the various dead and dormant cells.
Research has shown that microorganisms isolated from permafrost are similar to those of surface and aquatic cold ecosystems and habitats. Permafrost harbors also cyanobacteria, green alga, yeast, action- and micromictes, as well as biologically active macromolecules (Gilichinsky & Rivkina 2011). At subzero temperatures the rates of biochemical reactions and biological processes become extremely low, which ensures the preservation of organic material. The unfrozen water serves as a cryoprotector and permafrost communities can be defined as psychro-halo-tolerant microbes (Tiedje et al. 1994).
Permafrost harbors a very large number of viable bacteria, yet Yergeau et al. (2010) reported 20-fold fewer cells in permafrost compared to the active layer. The total number of microbial cells in permafrost is estimated to be 105-108 cells per gram dry mass, of which only 102-106 cells per gram dry mass are viable (Rivkina et al. 1998, Vishnivetskaya et al 2000).
2.3.3 Microbial activity
In natural systems, the action of soil microorganisms is a major determinant of efficient nutrient cycling. Soil microbial activity reflects microbiological processes of soil microorganisms, such as decomposition, and can often be estimated through the exchange rate of gases (Panikov 2009). The rate of CO2 production is commonly used as a measure of microbial activity in the soil, but other gases might be considered as well (Solaiman 2007). It is the potential indicator of soil quality, as plants rely on soil microorganisms to mineralize organic nutrients for growth and development (Chen et al. 2003).
2.3.3.1 Carbon dioxide (CO2)
Carbon enters the soil system mainly as dead organic material from plants and animals, but can also be fixed as CO2 (Clemmensen et al. 2013). CO2 is acquired from the atmosphere and converted into organic compounds in the process of photosynthesis in the presence of sunlight or chemosynthesis in the absence of sunlight (Madigan et al. 2012). The organic carbon is then used by microorganisms as energy source and broken down during decomposition, leading to the production of CO2 as a waste product that can be released back into the atmosphere. Thus the main source of CO2 from soils is soil respiration (Bargett et al. 2005).
16 2.3.3.2 Methane (CH4)
As already mention, the production of CH4 is called methanogenesis and occurs under oxygen limited conditions. Organisms capable of methanogenesis are methanogens. The only identified methanogens are part of the domain Archaea. These are strictly anaerobic and belong to the phylum Euryarchaetoa
(Pina et al. 2011). Most methanogens are autotrophic producers which can reduce CO2 with H2 to CH4
(Thauer et al. 2008) as the final step in the decay of organic matter. Nevertheless, CH4 can be consumed by methane-oxidizing bacteria called methanotrophs which belong to the gram-negative Proteobacteria. Oxidation of atmospheric methane occurs under the presence of oxygen in soils (Hanson & Hanson 1996).
2.3.3.3 Nitrous oxide (N2O)
+ The soil organic nitrogen is converted into the ion ammonium (NH4 ) through the process of + - ammonification. NH4 is then oxidized under aerobic conditions into the ion nitrite (NO2 ), producing - N2O as a byproduct, and further into nitrate (NO3 ) through the process of nitrification (Paul & Clark - 1996). NO2 oxidation is performed by ammonium oxidizing archaea (AOA) belonging to the phylum Crenarchaeota and ammonium oxidizing bacteria (AOB) belonging to the phylum Proteobacteria, mainly of the classes beta- and gammaproteobacteira.
- - Oxidation of NO2 to NO3 is primarily done by bacteria of the genus Nitrobacter belonging to the class alphaproteobacteria of the phylum Proteobacteria and Nitrospira belonging to the phylum Nitrospirae (Madigan et al. 2012). The degree to which nitrification happens decreases when pH - - decreases (Crutin et al. 1997). Under anaerobic conditions, NO2 and NO3 can further be reduced into gaseous nitrogen such as N2O and N2 through the process of denitrification (Verstraete & Focht 1977, Tiedje 1988).
Fungi are capable of nitrification and denitrification and often dominate the microbial biomass of temperate grassland soils. Many fungi though lack N2O reductase, so N2O is the final product of fungal denitrification (Shoun et al. 1992). This fact is of ecological significance, since N2O is a radiative active trace gas and N2 is not (Lauglin & Stevens 2002).
2.4 Climate change
2.4.1 Overall effect on the Arctic
Climate change refers to any significant change in the usual climate lasting for an extended period of time. This includes changes in global measurements of temperature, precipitation or wind patterns,
17 among others, that occur over several decades or longer. Global warming refers to the recent and ongoing rise in global mean temperature near Earth‟s surface, representing only one aspect of climate change. It is mostly caused by increased concentrations of greenhouse gases (GHGs) in the atmosphere (IPCC 2013, Martin & Hine 2015).
Climate change can have large and unpredictable effects on cold environments. Sattler et al. (2008) argue that these particular environments will vanish with rising temperatures and associated abiotic factors. At the same time liquid water availability will increase, which may, for some time, stimulating the productivity of adapted populations at the same time enhance biotic pressures, such as competition for resources.
Temperature seems to rule everything alive and consequently ecosystem processes and services on which organisms unconditionally depend. In the event of global warming, northern ecosystems are anticipated to undergo the most significant increase in surface temperature, with twice as high temperature increase as at mid-latitudes (Quinn et al. 2008) and the mean autumn-winter temperatures in the Arctic are projected to increase between 3 and 6 °C by 2080 (AMAP 2011). Drastic climate change could increase the depth of the soil‟s active layer and stimulate soil respiration thus increasing
CO2 production. In the warming scenario, northern ecosystems could cease to be a carbon sink and become carbon source instead. However, the net result of climate change on the cold carbon balance is difficult to predict, given that the loss of permafrost in the northern ecosystems may also increase the land area of more productive and warmer soils that may lead to further carbon sequestration (Lal 1995).
According to Roxy et al. (2014) a rise in soil temperature as a result of global warming will lead to changes in the soil heat flux, which represents the amount of radiant energy absorbed or released at the soil surface during a given time. This depends on the amount of solar radiation received by the surface and the moisture status of the soil. A change in the physical state of water from frozen to liquid due to rising air temperatures will make water available for microorganisms for biological processes, stimulating further production of CO2.
Temperature and precipitation mainly govern the rate of chemical reactions, growth and activities of soil biota. Climate change will not just lead to the increase of temperature, but also in the increase of precipitation, mainly as snowfall (increase of 15-30 % with greatest impact expected in Siberia) (AMAP 2011). Snow has an insulating effect on soil. With increased depth of snow coverage, the soil will be further heated, resulting in thickening of the active layer and permafrost degradation (Zhang et al. 2008, Schuur et al. 2009). At the same time snow will tend to lie on the ground for 10-20 % less time each year in most of the Arctic, due to earlier melting in spring as a result of global warming (AMAP 2011), consequently leading to an earlier start of the growing season (Elberling et al. 2013).
18 2.4.2 Greenhouse gases & their warming potential
Gases that trap heat in the atmosphere are called greenhouse gases (GHGs). They warm the Earth through the greenhouse effect (GHE) by absorbing energy and slowing the rate at which the energy escapes to space. Different GHGs have a different GHE depending on their ability to absorb energy (called their radiative efficiency) and how long they are in the atmosphere (also known as lifetime). We compare GHGs by their Global Warming Potential (GWP) (Allaby 2015). This method is a measurement of how much energy the emission of 1 ton of a gas will absorb over 100 years, relative to the emissions of 1 ton of CO2. The larger the GWP, the more that a given gas warms the Earth compared to CO2 (Tveit et al. 2013, Nelissen et al. 2014).
Besides CO2, the primary atmospheric trace gases contributing to the enhanced global warming are
CH4 and N2O. They have a shorter lifetime (12 and 114 years, respectively CH4 and N2O) compared to
CO2 (up to 200 years) (CDIAC 2011), but they have greater GWP than CO2 (about 25x and 298x, respectively) (Knoblauch et al. 2013). Increased atmospheric concentrations of these GHGs due to global warming would thus lead to further increase of the global mean temperature, creating a powerful positive feedback mechanism (see figure 2) (Heimann & Reichstein 2008).
Figure 2: The positive feedback loop established by global warming. Increased air temperature will warm up the ground thus leading to permafrost thaw and the release of nutrient. Increased availability will stimulate microbial decomposition thus leading to the production of further GHGs that are release to the atmosphere and enhancing the effect of global warming. Modification of the loop provided by Centre d‟études Nordiques.
GHGs are actively produced through microbial decomposition, both within the active layer that undergoes seasonal thaw, but also at sub-zero temperatures inside the permafrost (Rivkina 2000, Panikov & Sizoya 2007). Gases produced inside permafrost may be trapped and unable to diffuse in the presence of ice functioning as a barrier (Brook et al. 1996, Mackelprang et al. 2011). When permafrost thaws, these trapped gases are released in addition to that the stored organic material becomes available for microorganisms, stimulating activity, thereby increasing the amount of GHGs emitted to the atmosphere (Schuur et al. 2009, Elberling et al. 2010, Elberling et al. 2013).
19 2.4.3 Global warming’s effect on permafrost-affected soils
Climate model projections indicate a reduction of 31 – 81% in the upper 3.5 m of permafrost at high northern latitudes by the end of the 21st century (IPCC 2013). Several studies have already projected an increase in the depth of active layer about 10-40 cm within the next 70 years (IPCC 2007, Daanen et al. 2011, Hollesen et al. 2011) that will include layers that have not been thawed within the past few hundred years. They also show high concentrations of carbon and nitrogen in the top active layer and decreasing concentrations with depth (Elberling et al. 2010). Generally, the ice content in permafrost is considerably higher than the water content in the active layer. By the end of the 21st century, an increase of mean annual ground temperature by up to 6 °C is expected in addition to deepening of the active layer by up to 2 m in East Siberia (Stendel et al. 2008).
As already mention, permafrost affected soils harbor large carbon stores. Even though the active layer may undergo seasonal thawing that allows microbes to access some of the soil stores, permafrost thawing may release additional enables microorganisms to access these stores (Kvenvolden & Lorensen 1993). Increased atmospheric concentrations of GHGs are very likely to have a larger effect on climate in the Arctic than anywhere else on the globe. This increases the risk of losing soil carbon due to acceleration in decomposition of organic soil matter under warming climate (AMAP 2011).
Christensen et al. (2004) showed that permafrost thawing in northern Sweden has resulted in land- cover changes and led to a net increase in the CH4 emission from the soil to the atmosphere. In general, there is a top down mobilization of carbon in soils and by freezing of the ground carbon will be sealed inside of it. Carbon can be trapped as organic compounds as well as inorganic ones, such as gases. Permafrost soils contain nearly twice as much carbon as the atmosphere. As a result of thawing, the release of trapped carbon leads to accumulation of CO2 and CH4 in the atmosphere, thereby removing large quantities of carbon from the terrestrial environment (Rivkina 2007, Schuur et al. 2009). Today, melting of permafrost and the creation of runoff streams will increase the loss of particulate organic carbon to arctic rivers, thereby increasing the supply of organic matter towards coastal sediments (Guo et al. 2007).
Climate models forecast indicate that continued warming will increase the rates of permafrost thawing at the poles (Koven et al. 2011, Schäfer et al. 2011). Increased arctic surface temperatures cause a particular susceptibility of Arctic permafrost to degradation. Global warming models predict 0-15% increase in the depth of seasonal thawing over most of the permafrost area by 2025 and 30 % and more by 2080 (Anisimov et al. 2007).
Climate change may lead to progressive modification of the permafrost thermal regime, and hence its physical properties and distribution. The rates and magnitudes of changes are difficult to predict. The key properties of interest in permafrost studies are temperature and ice content (Harris et al. 2008).
20 The presence of saline pore waters leads to freezing point depression and affects the soils geophysical properties.
Thaw has two important geomorphic consequences: (1) a reduction in soil strength due to the change to an unfrozen state, and (2) a reduction in soil volume (consolidation) due to the loss of excess ice (French 2007). As the active layer deepens, thaw consolidation produces melt-out horizons and may trigger soft-sediment deformation.
Global warming at high latitudes is putting large areas of ice-rich permafrost at risk of thermokarst activity and related disturbances (Murton 2009). Thawing and mixing of soil destroys the physical structure of permafrost including the osmotic gradients in water films, replacing the terrain with very irregular surfaces of marshy hallows and small hummocks, offering suitable conditions for anaerobic mineralization of organic matter and hence microbial methanogenesis (Nelson 2003, Payette et al. 2004, Christensen 2004).
2.4.4. Global warming’s effect on microorganisms
As a group, microorganisms are highly mobile, can tolerate most environmental conditions, and have short generation times that can facilitate rapid adaptation to change. Although extreme environmental conditions restrain the metabolic activity of arctic microbes, they preserve huge potential that is likely to display the same activity as boreal analogues during climate warming (ACIA 2004). Microbial communities in permafrost soils respond to variations in soil environments and are particularly affected by soil moisture, pH and available organic material (Mannisto et al. 2007, Wallenstein et al. 2007, Chu et al. 2011). Therefore it has been suggested, that there is a close association between the microbial community and soil characteristic in response to changes in climate.
Decomposition of organic matter in freshly thawed permafrost depends on the interactions between carbon quality, abiotic conditions, geomorphology and decomposer activity, which depends on functional traits of microbial community that are often shaped by substrate availability (Schädel et al. 2014, Treat et al 2015). Functional potential of permafrost microbial communities is likely to have a large effect on the decomposition of permafrost and carbon loss to the atmosphere, but this effect is difficult to predict with current knowledge (Graham et al 2011). Understanding the functional traits of permafrost microbial community may improve our predictions of decomposer activity and potential GHG flux from permafrost after thaw.
Episodic warming events, associated permafrost thaw and high flows have caused inundation of previously dry soils. An increase in moisture alters the conditions for soil microbes through the mobilization of nutrients and salts, thereby stimulating primary production and carbon input to soils (Simmons et al. 2009). However, climate change is likely to alter microbial community composition
21 and substrate utilization (Lipson et al. 2009). Global warming will result in a significant reduction in evenness of bacterial community, but at the same time increase the microbial activity. Increased growth rate and use of substrates result in faster carbon turnover in soils, leading to a decline in accumulated organic carbon (Deslippe et al. 2012).
22 3 MATERIALS AND METHODS
3.1 Field site
A soil core was collected on July 25th, 2014 near Brønlundhus in Peary Land, Greenland (GPS coordinates N 82° 10.276; W 30° 30.446) using a motorized hand drilling equipment consisting of a Stihl drilling engine, an expandable dill string and a 40 cm long cylinder drill head (see figure 3). Samples were packed in plastic bags in the field and immediately stored in a freezer box, while they were kept below -5 °C at the Research Station and during transport, until stored at -6 °C in the laboratory at the Center for Permafrost (CENPERM), Copenhagen.
Figure 3: The motorized hand drilling equipment is being brought into position for drilling. The surface soil in Peary Land shows no vegetation, only rock coverage. Picture taken and provided by Bo Elberling.
23 3.2 Soil core
The core has a total depth of 350 cm and was divided into intervals of 10 cm, except for the depth 150- 170 cm, which has a 20-cm interval. The depth intervals 10-20 cm, 30-40 cm and 130-140 cm are lacking for unknown reasons, resulting in a total of 31 intervals. To ensure that the permafrost core remains uncontaminated, all instruments used to split the core were cleaned with 70 % ethanol. The intervals will further on be referred to as depths.
Prior the incubation experiments, the individual depths were transported to a 10 °C room to thaw the outer parts of the samples. Using sterile scalpels, the outer layer of the samples (approximately 1 cm) was scraped off and set aside for analyses of soil properties. The inner part of the core was placed in a sterile bag and homogenized by placing the sterile bags on sterile aluminum foil on a 60 cm2 metal plate and crushing the still frozen sample gently with a hammer (see figure 4). Afterwards the soil was sieved through an 8-mm sieve to remove bigger stones from the samples. The homogenized soil was used for the incubation experiments and DNA isolation.
Figure 4: A sterile bag filled with soil sample placed on top of sterile aluminum foil on a metal plate. The aluminum foil was wrapped around the sterile bag and gently hit with a hammer to homogenize the sample. The metal plate was cleaned with ethanol between each sample and the foil was switched between each sample as well.
24 3.3 Incubation experiments
Microbial activity in incubated samples was measured through the production of CO2, CH4 and N2O.
3.3.1 Aerobic incubation
For the aerobic incubation, 8 g homogenized soil from each depth was placed in five 100-mL serum bottles each, resulting in five replicate incubations per depth. The bottles were closed using 1-cm thick butyl rubber septa and aluminum caps. The serum bottles were incubated for six months at 2 °C in the dark and never opened nor flushed.
3.3.2 Anaerobic incubation
The anaerobic incubation was set up similar to the aerobic incubation except that the closed serum bottles were flushed for 2 minutes with nitrogen 5.0 at a speed of ~100 mL/min to create an anoxic atmosphere. To sustain this atmosphere, all bottles were flushed with nitrogen every four weeks using a cobber column (see figure 5). The serum bottles were incubated for six months at 2 °C in the dark as well.
(a) Figure 5: The samples were coupled to a multi-flusher (a) which was coupled to a cobber column (b). The column was connected to the nitrogen 5.0.
(b)
25 3.3.3 Sampling & measurement of produced gas
In order to obtain an estimate of the microbial activity, sampling from the serum bottles was performed over five days every 8th week with 48-72 hours between each sampling. Prior to each sampling, 17 mL of laboratory air and nitrogen, respectively aerobic and anaerobic incubation, were added to compensate for the pressure difference through the gas extraction over the following 168 hours. Gas samples were always collected after 0, 48, 72, 120 and 168 h, with 0 h being 24 hours after the flushing of the anaerobic incubation and the addition of the 17 mL to both incubations to ensure re-equilibration of the atmosphere.
Gas collection was done by extracting 3 mL from the head space of each bottle using a 5-mL syringe and 0.4-mm needles (see figure 6). The extracted samples were filled into 3-mL exetainer vials (LABCO, Lampeter, UK) and analyzed using an autosampler Figure 6: 3 mL of head space gas (Mikrolab Aarhus, Højbjerg, Denmark) and 7890A GC system was extracted from each serum (Agilent Technologies, Glostrup, Denmark) equipped with a flame bottle using a 5 mL syringe and ionization detector and an electron capture detector. The GC 0.4-mm needle. The same needle and syringe were used for analysis gave the amount of gas produced as ppm. The production replicates, but renewed between rate was calculated as ng gas produced per g dry soil per day. samples.
3.4 Soil properties
The analyses of the soil properties were conducted on the outer part of the soil core. The measurements of total organic carbon (C), total organic nitrogen (N), total phosphorous (P), soil moisture and conductivity were performed on the soil prior incubation only, while measurements of + - ammonium (NH4 ), nitrate (NO3 ), dissolved organic carbon (DOC) and soil pH were also conducted on the incubated soil. For every analysis, three technical replicates were produced for each depth.
+ - 3.4.1 NH4 , NO3 , DOC & pH
+ - For the analysis of NH4 , NO3 and DOC in the non-incubated soil, 10 g soil from each depth was diluted with 50 mL ddH2O and shaken at 200 rpm for 60 minutes at room temperature; afterwards each diluted sample was centrifuged at 4300 rpm for 10 minutes and filtered through a G/D filter
26 (Whatman™, Maidstone, UK). For the same analysis in the incubated soil, 40 mL ddH2O were added to the 8 g soil inside the serum bottles, but else treated the same way as the non-incubated soil.
The extracts were filled into two 20-mL plastic bottles per depth. One bottle was used for the + - measurement of NH4 , NO3 and DOC and stored at -18 °C until needed, while the other bottle was + - used for pH analysis straight away. NH4 and NO3 concentrations were determined by flow injection using a FIAstarTM 5000 (FOSS, Hillerød, Denmark) following manufacturer‟s instructions. DOC was analyzed using the TOC-L 00209/TNM-L 00207 (Shimadzu, Kyoto, Japan). The pH was measured using the MeterLab® PHM222 and PHM240 (Radiometer, Copenhagen, Denmark).
3.4.2 Conductivity
An additional 2 g from each depth were diluted with 10 mL double distillated water (ddH2O) and shaken at 200 rpm for 30 minutes, afterwards centrifuged at 4300 rpm for 10 minutes and decanted into test tubes for the measurement of the conductivity using the MeterLab® CDM230 (Radiometer, Copenhagen, Denmark).
3.4.3 Soil moisture, total C, total N & total P
A further 5 to 10 g from each depth were placed in paper bags, weighted, burned at 70 °C for 48 hours and weighted again for calculation of the soils water content based on the weight difference. The dried soil was then crushed using a Pulverisette 23 (FRITSCH, Idar-Oberstein, Germany) and stored in 3- mL glass bottles until needed for the measurement of total C, total N and total P.
Before the measurement of total P content, the soil was degraded. To do so, 50 mg of each sample was placed inside a reagent tube. 5 mL of concentrated sulfuric acid (H2SO4) and 1 mL hydrogen peroxide
(H2O2) were added and left over night. The next day all samples were heated to 400 °C for 1 h.
Afterwards, the reagent tubes were filled to a volume of 25 mL with ddH2O and carefully shaken. The solution was transferred in to 20-mL plastic bottles. Remaining solution was properly disposed. Total P concentrations were determined by flow injection using a FIAstarTM 5000 (FOSS, Hillerød, Denmark) following manufacturer‟s instructions.
Total organic C and total organic N were analyzed by weighting between 250 g and 300 g dried, pulverized soil into tin foil filters and analyzing those samples on a TrueSpec CN determinator (LECO Corporation, St Joseph, USA), following manufacturer‟s instructions.
27 3.4.4 Temperature
In situ temperature was measured using standard temperature loggers (CAMPBELL Scientific Inc., Lougborough, UK). After the core was taken on July 24th, 2014, the bore hole was equipped with sensors at the depths 0, 5, 15, 25, 45, 95, 145, 195, 245 and 345 cm. Data were collected on an hourly basis from July 25th, 2014 until March 30th, 2016.
3.5 Molecular analyses
Due to the limited laboratory time within the timeframe of this thesis the number of analyses had to be shortened and concentrated to the most important in relation to the thesis. Therefore all molecular analyses were done on the samples prior to the incubation experiment to minimize the amount of data.
3.5.1 DNA isolation
Two 2-mL SC Micro PCR-PT tubes (Sarstedt AG & CO, Nümbrecht, Germany) were filled with approximately 2 g homogenized soil for each depth and freeze-dried at -80 °C. From this dried soil DNA was isolated using the Power Soil DNA extraction kit (Mo Bio Laboratories, Carlsbad, USA) following manufacturer‟s instruction until the last step, where the isolates were only diluted with 50 µL of solution C6.
Two technical replicates were produced for each tube, resulting in a total of four technical replicates per depth for molecular analysis resulting in a total of 124 samples.
3.5.2 PCR test run
PCR was done for the amplification of the prokaryotic 16S ribosomal RNA (rRNA) gene and the fungal Internal Transcribed Spacer 2 (ITS2) region to test for the presence of these genes. The tested samples corresponded to DNA isolates from samples at 20-30 cm, 70-80 cm, 140-150 cm, 170-180 cm, 220-230 cm and 290-300 cm depth.
A master mix for one 30 µL reaction consisted of 0.3 µL HIFI Polymerase, 6 µL reaction buffer (PCR Biosystems, London, UK), 3 µL bovine serum albumin (BSA; BIORON GmbH, Ludwigshafen, Germany), 0.6 µL dNTP (10 mM), 1.5 µL forward primer, 1.5 µL reverse primer and 12.1 µL clean PCR water. The bacterial 16S rRNA region was targeted by EUB518P (5‟ ATT ACC GCG GCT GCT GG 3‟) (Fierer et al. 2005) and EUB341F (5‟ CCT ACG GGA GGC ACA AG 3‟) (Muyzer et al. 1993) primers, while fungal ITS2 region were targeted by ITS4 (5‟ TCC TCC GCT TAT TGA TAT GC 3‟) (White et al. 1990) and ITS7 (5‟ GTG ART CAT CGA RTC TTT G 3‟) (Ihrmark et al. 2012)
28 primers. All primers were diluted 10x. The master mix was filled into 0.2 mL PCR tubes and 5 µL DNA template was added. Subsequently, each sample was divided into 3x10-µL reactions to ensure a high concentration of gene copies.
Thermal conditions for 16S amplification were 95 °C for 1 min, followed by 30 cycles of 95 °C for 15 sec, 50 °C for 20 sec and 72 °C for 20 sec, ending with 72 °C for 5 min. The previously divided PCR products were collected in a single tube and checked by gel electrophoresis at 100 V for 45 minutes.
3.5.3 qPCR
Microbial gene abundance was estimated by qPCR using 20 µL reactions on CFX96 Touch qPCR system (Bio-Rad, Richmond, USA) targeting 16s rRNA and ITS2 gene copies (henceforth mentioned as 16S and ITS). An Escherichia coli K-12 strain was used as standard for 16S, with reported seven 16S rRNA genes per genome (Blattner et al., 1997). For the ITS standard, a pUC18 plasmid (SanpGene GSL Biotech, Chicago, USA) was used. The plasmid‟s host was a One Shot® TOP10 Chemically Competent E. coli (Thermo Fisher Inc., Waltham, USA) and was purified using the QIAprep Spin Miniprep Kit (Qiagen, Venlo, The Netherlands) following manufacturer‟s instructions.
A master mix for 100 reactions consisted of 200 µL BSA, 80 µL forward primer, 80 µL of reverse primer, 540 µL of clean PCR water and 1000 µL SyBR Green Lo-Rox polymerase (PCR Biosystems, London, UK). The primers were the same as mentioned previously. The master mix was divided on a 96-well plate (Thermo Fisher Inc., Waltham, USA) resulting in 19 µL in each well. To each well 1 µL DNA template was added. The qPCR reactions were run in technical triplicates.
Thermal conditions for 16S amplification were 95 °C for 2 min, followed by 40 cycles of 95 °C for 10 sec, 55 °C for 30 sec and 72 °C for 30 sec, followed by 72 °C for 6 min and ended with 55 °C for 10 seconds followed by an increase of 0.5 °C every 10 seconds until 98 °C was reached (see figure 7). Thermal conditions for ITS were similar, except for the annealing temperature, which was 56 °C instead of 55 °C (steps 3 and 7 on figure 7).
Figure 7: Thermal conditions for 16S qPCR. Thermal conditions for ITS qPCR were similar, except for step 3 and 7, which started at 56 °C.
29 3.5.4 Illumina MiSeq sequencing
PCR preparations were as mentioned in section 3.5.2 (PCR test run), thus with different primers. The primers were diluted 10x and mixed, based on 12 tagged forward primers and 12 tagged reverse primers, according to a matrix resulting in 144 primer combinations, of which 131 were used (see Appendix 1). Due to this, each sample was tagged and could be identified later on in the sequence data. PCR was conducted on all 124 samples and 7 negative controls to amplify the 16S rRNA gene. Five runs were conducted, four with 28 samples and 1 negative control each, one with 12 samples and 3 controls.
Each PCR was tested by gel electrophoresis at 100 V for 45 minutes. Weak bands were excluded and PCR repeated using 35 cycles instead of 30 and PCR products checked by gel as mentioned above. Afterwards, the PCR products were purified using PCR Clean up (CleanNA, Alphen aan den Rijn, The Netherlands) following manufacturer‟s instructions. The DNA concentration of each sample was measured using Qubit® dsDNA HS Assay Kit (Thermo Fisher Inc., Waltham, USA) following manufacturer‟s instructions by adding 3 µL sample.
PCR products from the first 68 samples were combined in one 2-mL Eppendorf tube while the remaining 63 PCR products were combined in another tube. The DNA concentration of both tubes was measured using Qubit®. The tubes were then vacuum-centrifuged at 30 °C for 1200 rpm for 3 hours to remove all liquids. The attempt was to attain 20 ng DNA per µL in each tube. Due to too high concentrations, the centrifuged samples were diluted to an end volume of 200 µL with clean PCR water. The DNA concentrations were checked again using Qubit®.
Ligation of Illumina adaptersequences was performed using the MiSeq Reagents kit (Illumina Inc., San Diego, USA) following a modified version of the manufacturer‟s instructions by Jana Vorískova (data not published). Due to too low concentrations of the ligation end product, the product was amplified in a ten-cycle PCR using 10 µL buffer, 0.5 µL HIFI polymerase (PCR Biosystems, London, UK), 2 µL primer I5 (5ʹ AAT GAT ACG GCG ACC ACC GAG AT 3ʹ), 2 µL primer I7 (5ʹ CAA GCA GAA GAC GGC ATA CGA 3ʹ) (Illumina Inc., San Diego, USA), 30.5 µL clean PCR water and 5 µL sample for one reaction. The PCR was run on a 2720 thermal cycler (Applied BiosystemsTM, Foster City, USA) under the conditions 95 °C for 1 min, 10 cycles of 95 °C for 15 sec, 56 °C for 15 sec and 72 °C for 30 sec, followed by 72 °C 5 min and a hold on 4 °C. The library DNA was purified using the AMPure XP beads (Beckman Coulter Inc., Brea, USA) and concentrated using the DCCTM-25 (Zymo Research, Irvine, USA), both following the manufacturer‟s instructions. The samples were sequenced on an Illumina MiSeq sequencer using the V2 kit (Illumina Inc., San Diego, USA) following the manufacturer‟s instructions at the University of Copenhagen.
30 3.6 Data analysis
Sequencing data were analyzed and statistically tested through the application of ANOSIM (ANalysis Of SIMilarity) using Primer 7 (Primer-E, Aukland, New Zealand). All remaining data were analyzed using Microsoft Office Excel 2007 (Microsoft, Redmond, USA) and Gnuplot 2010 (Geeknet Inc., Fairfax, USA).
The F-test was used to determine what type of t-test should be applied at a significant level of p < 0.05. Student‟s t-test for dependent data was used to test for a possible significant difference between + - the concentrations of DOC, NH4 , NO3 and pH in the incubated and non-incubated soil. Student‟s t- test for independent data was used to test for significant differences of soil properties, GHG production and the number of 16S rRNA gene copies between different soil layers. Spearman‟s rank correlation was used to determine a significant correlation between soil properties and CO2 production.
31 4 RESULTS
4.1 Soil properties
4.1.1 Temperature
The mean surface and soil temperatures for each month of the year 2015 are illustrated on figure 8. Thaw of the AL began on June 5th at the surface at 1.28 °C and increased with depth. The active layer refroze on September 1st. Figure 9 shows the temperature at the 95 cm for the months July, August and September. In July the temperature increased towards 0 °C. In August the temperature got closer to 0 °C, but never above. The closest temperature was -0.46 °C measured on the 19th and in September the temperature decreased again. The mean temperature for August was -0.59 °C (+/- 0.1).
Figure 8: The mean temperature for each depth from each month of the year 2015. The green stripes represent the active layer and the blue stripes represent the permafrost.
32
Figure 9: The temperature at 95 cm for the months July (blue), August (red) and September (green). In July it can be seen that the temperature increases towards 0 °C. In August the temperature comes close to 0 °C, but never above and in September the temperature decreased again.
33 4.1.2 Total C, total N & total P
Figure 10 shows the total amount of C (%), N (%) and P (ppm). The amount of total N was significant different (p < 0.01) between the upper and middle soil layer as well as between the lower and middle soil layer. Derived from this pattern I was able to define three different layers within the soil core: an active layer (AL) ranging from 0-100 cm, a young permafrost (YP) layer ranging from 100-200 cm and an old permafrost (OP) layer ranging from 200-350 cm.
The average amount of total N is 0.04 (+/- 0.01) % inside the AL, 0.1 (+/- 0.01) % in the YP and 0.05 (+/- 0.02) % in the OP, indicating a significant higher amount of total N present in the YP compared to the layers above and below. The average amount of total C is 3.3 (+/- 0.7) % in the AL, 3.4 (+/- 0.9) % in the YP and 3.6 (+/- 0.7) % in the OP indicating an increase of total C with depth, but no clear pattern can be seen as for total N. The average amount of P is 330 (+/- 39) ppm in the AL, 342 (+/- 39) ppm in the YP and 374 (+/- 57) ppm in the OP indicating a slight, but not significant increase.
Figure 10: The amount of total N (left), total C (middle) and total P (right). While total N shows a clear difference between the soil layers, total C and P show no difference. Bars indicate standard error of the mean.
34 4.1.3 Conductivity & soil moisture
The soils conductivity and moisture are illustrated on figure 11 with a conductivity average of 776 (+/- 806) mS in the AL, 1750 (+/- 668) mS in the YP and 2377 (+/- 498) mS in the OP. Conductivity is low within the AL but increases significantly (p < 0.05) with depth. The average water content in the AL is 23 (+/- 22) % of dry weight soil, 40 (+/- 4) % of dry weight soil in the YP and 36 (+/- 7) % of dry weight soil in the OP. The water content is low inside the AL but increases towards the YP, but no significant difference can be seen between the two permafrost layers.
Figure 11: The conductivity [mS] (left) and water content [% of dry weight soil] (right) of the soil core. Bars indicate standard error of the mean.
35 4.1.4 pH
Figure 12 shows the soil pH before and after the incubation experiment. The pH before incubation was slightly alkaline, ranging from 8.37 at the surface to 8.16 at the bottom of the soil core. The average pH within the AL was 8.48 (+/- 0.09), 8.52 (+/- 0.1) in the YP and 8.31 (+/- 0.07) in the OP, showing an increase within the AL towards YP and decrease within the permafrost. The overall pH after the experiment did not significantly change, ranging from an 8.25 at the soil surface to an 8.05 at the bottom of the soil core. There is a great drop within the AL after the experiment from 20-30 cm down to 80-90 cm depth. The average pH within the AL was 8.15 (+/- 0.09), 8.11 (+/- 0.04) in the YP and 8.08 (+/- 0.04) in the OP, showing an increase within the AL towards the YP as well, but difference within the permafrost. The difference between the soil layers was significant between the AL and OP after incubation and the YP and OP both before and after incubation. The significant level for pH was p < 0.05.
Figure 12: The soil pH before (blue ♦) and after (green ●) the aerobic incubation experiment. Bars indicate standard error os the mean and significant difference (p < 0.05) between the samples is indicated with an * at the sight side.
36 4.1.5 DOC
The DOC concentrations are low, but a significant increase in the OP at depths 220-240 cm and 260- 300 cm after soil incubation was observed (figure 13). The concentrations range from 0.02 µg g-1 dry soil at the soil surface to 0.03 µg g-1 dry soil at the bottom of the soil core before incubation, and 0.03 µg g-1 dry soil at the surface to 0.02 at the bottom after incubation, indicating no significant difference. Before incubation, the average concentrations found in the AL was 0.09 (+/- 0.07) µg g-1 dry soil and 0.08 (+/- 0.03) µg g-1 dry soil in the YP and the OP. After incubation, the average concentrations found in the AL was 0.03 (+/- 0.02) µg g-1 dry soil, 0.03 (+/- 0.02) µg g-1 dry soil in the YP and 0.45 (+/- 0.88) µg g-1 dry soil in the OP. The level of significant difference in DOC concentrations in the soil before and after incubation was p < 0.05.
Figure 13: The concentration of DOC before (blue ♦) and after (green ●) soil incubation. Standard error bars are included and significant difference (p < 0.05) between DOC concentration before incubation and after incubation is indicated with an * at the sight side.
37 - 4.1.6 NO3
- NO3 concentration was significantly lower in the incubated soil compared to the non-incubated soil (figure 14). The concentration range from 3.9 µg g-1 dry soil at the soil surface to 0.1 µg g-1 dry soil at the bottom of the soil core before incubation, and 5.4 µg g-1 dry soil at the surface to 0.02 at the bottom after incubation. Before incubation, the average concentrations found in the AL was 1.5 (+/- 1.2) µg g-1 dry soil, 0.4 (+/- 0.3) µg g-1 dry soil in the YP and 0.2 (+/- 0.2) µg g-1 dry soil in the OP. After incubation, the average concentrations found in the AL was 1.2 (+/- 2) µg g-1 dry soil, 0.07 (+/- 0.07) µg g-1 dry soil in the YP and 0.05 (+/- 0.04) µg g-1 dry soil in the OP. These results show a - significant decrease of NO3 concentration with depth. A significant difference between soil layers was only seen in the non-incubated soil between the AL & YP and AL & OP. The level of significance was p < 0.05.
- Figure 14: The concentration of NO3 before (blue ♦) and after (green ●) soil incubation. Bars indicate standard error of the mean and significant difference (p < 0.05) between the samples is indicated with an * at the sight side.
38 + 4.1.7 NH4
+ The concentration of NH4 increased significantly with depth, both before and after soil incubation (figure 15). The concentrations range from 0.3 µg g-1 dry soil at the soil surface to 6.1 µg g-1 dry soil at the bottom of the soil core before incubation, and from 0.2 µg g-1 dry soil at the surface to 3.7 µg g-1 dry soil at the bottom after incubation. Before incubation, the average concentrations found in the AL was 0.7 (+/- 1.1) µg g-1 dry soil, 3.7 (+/- 1) µg g-1 dry soil in the YP and 6.1 (+/- 1.7) µg g-1 dry soil in the OP. After incubation, the average concentrations found in the AL was 0.2 (+/- 0.2) µg g-1 dry soil, 2 (+/- 1.7) µg g-1 dry soil in the YP and 5.9 (+/- 3.1) µg g-1 dry soil in the OP. Not all samples showed a significant difference in concentration before and after incubation. A significant difference in concentration between soil layers was obtained between the AL & YP and AL & OP both before and after incubation whereas a difference between YP and OP was only obtained before. The level of significance was p < 0.05.
+ Figure 15: The concentration of NH4 before (blue ♦) and after (green ●) soil incubation. Bars indicate standard error of the mean and significant difference (p < 0.05) between the samples is indicated with an * at the right side.
39 4.2 Incubation experiments
4.2.1 Aerobic incubation
The flux for each month of sampling resulted in mainly negative slopes (data not shown). Since the cap on the serum bottles was never removed, a total flux for the overall period was calculated based on the gas samplings from the first day of each sampling month (see figure 16). These results gave an estimate of microbial activity and were the focus from here on.
CO2 production was lowest in the active layer but increased highly significant with depth (p < 0.01). A significant difference in production between soil layers was obtained between the AL and YP as well + as AL and OP. Production was also significantly correlated to the concentration of NH4 and soil moisture (p < 0.01) and conductivity (p < 0.02).
CH4 and N2O fluxes were very small; CH4 production is highest at the depth 90-100 cm and N2O production was first observed in the lower permafrost at 200 cm depth. N2O production was highly significant different (p < 0.01) between the YP and the OP.
4.2.2 Anaerobic incubation
The microbial activity under anaerobic conditions was little to non-existing for the production of CH4 and N2O, while the production of CO2 in month 0 at the beginning of the experiment had a negative production rate in soils from the depths 0-200 cm and a positive production rate in soils from 200-350 cm, but also non-existing in the following months (data not shown). Due to the lack of production, I excluded all data from the anaerobic incubation from further analyses and focused on the data obtained from the aerobic incubation.
40
Figure 16: The production rate of CO2 (left), CH4 (middle) and N2O (right) as ng gas produced per g dry soil per day. For
CO2, the production rate at the depths 0-10 cm, 20-30 cm, 50-60 cm and 60-70 cm are negative. For N2O, the production rate at the depths 40-100 cm, 120-130 cm, 140-150 cm and 190-200 cm are negative as well.CH4 shows only positive production rates. Bars indicate standard error of the mean.
41 4.3 Molecular analysis
4.3.1 PCR test run
Figure 17 shows the PCR products after gel electrophoresis for 16S (a) and ITS (b). The PCR products for 16S were of the same size (300 bp), while ITS could not be amplified. Therefore, I moved forward with Illumina MiSeq sequencing only for 16S.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
(a)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
(b)
Figure 17: The PCR product amplification of 16S rRNA gene (a) and ITS2 gene (b). The numbers correspond to the wells. In (a) the 100 base pair (bp) ladder was placed in well 2 and 30, while the samples were placed from well 4 to 27 in the order 20-30 cm, 70-80 cm, 140-150 cm, 170-180 cm, 220-230 cm and 290-300 cm. All four replicates were tested and placed in the order R1-R4 (replicate one to four). The negative control was placed in well 28 and the positive control was placed in well 29. In (b) the 100 bp ladder was placed in well 1 and 30, while the samples were placed in the wells 3 to 26 in the same order as in (a). The negative control was placed in well 27 and the positive control was placed in well 28. The thicker band on the ladder (red in (a) and bright in (b)) marks the 2x 500 bp fragment size.
42 4.3.2 qPCR
The qPCR efficiency was between 112.2 % and 205.0 % for 16S and between 173.9 % and 253.5 % for ITS. Even though I couldn‟t amplify the ITS2 gene through PCR, I did qPCR for the gene for a quantitative confirmation that no fungi were present (data not shown). A total of 1.4E+09 16S rRNA genes were amplified with lowest abundance in the YP and highest in the OP, but here the variance between samples was also greatest. The distribution of 16S rRNA genes throughout the soil core is shown on figure 18.
Figure 18: The 16S rRNA gene distribution on a logarithm scale. Standard error bars are included showing high variance between the measured samples in the lower OP.
43 4.3.3 Illumina MiSeq sequencing
A total amount of 10.551.985 sequences were acquired and identified from the soil core. The average number of depth was 84.892 (+/- 55.859). A highly significant difference in the number of sequences was detected between all three layers at a significant level of p < 0.001. 20.39 % of the sequences were found within the AL and 21.31 % in the YP, whereas 58.07 % of the sequences were found in the OP indicating an increase in number with increasing depth. However, by looking at figure 2A (Appendix 3), there is no clear tendency for the number of sequences to actually increase with depth. The highest number of sequences was found in at 320-330 cm depth. A multidimensional scaling (MDS) plot was used for the visualization of the bacterial diversity distribution throughout the soil core (figure 19).
Proteobacteria is the overall dominant phylum within this soil core; 32.74 % of the sequences belong to this phylum, followed by Actinobacteria with 24.29 %. Bacteria belonging to the phyla Firmicutes, Chloroflexi and Bacteroidetes were found in less abundance, representing 11.19 %, 8.95 % and 5.69 % of the total number of sequences, respectively. Actinobacteria is the only bacterial phylum that shows a clear tendency to decrease in abundance with increasing depth, whereas Firmicutes is the only phylum showing the opposite. Proteobacteria, Acidobacteria and Bacteroidetes show highest abundance in the YP, whilst Chloroflexi show higher abundance in the AL and Gemmatimonadetes‟ abundance is highest in the OP. Only 0.38 % of the sequences identified archaea, mainly belonging to the phylum Crenarchaeota (0.30 %), but a few belonging to the phylum Euryarchaeota were also found (0.08 %), which do increase in abundance with increasing depth as well. A total overview of all phyla present in the three layers can be seen on table 1.
44 Table 1: Overview of all phyla identified in the different layers of the soil core; the percentages are based on the total number of sequences for each soil layer.
Domain Phylum AL YP OP Archaea Crenarchaeota 0.50% 0.52% 0.15% Archaea Euryarchaeota 0.03% 0.09% 0.10% Bacteria Acidobacteria 1.88% 3.42% 1.99% Bacteria Actinobacteria 35.88% 25.47% 20.21% Bacteria Armatimonadetes 0.15% 0.37% 0.10% Bacteria Bacteroidetes 4.84% 9.43% 4.73% Bacteria Chlamydiae 0.06% 0.21% 0.36% Bacteria Chlorobi 0.11% 0.15% 0.04% Bacteria Chloroflexi 12.60% 6.14% 8.85% Bacteria Cyanobacteria 1.43% 0.86% 0.60% Bacteria Deinococcus-Thermus 1.55% 1.12% 1.09% Bacteria Elusimicrobia 0.03% 0.14% 0.03% Bacteria Firmicutes 4.14% 7.32% 15.30% Bacteria Fusobacteria 0.04% 0.04% 0.02% Bacteria Gemmatimonadetes 4.28% 2.49% 5.70% Bacteria Nitrospirae 0.05% 0.20% 0.04% Bacteria Planctomycetes 1.87% 2.89% 6.16% Bacteria Proteobacteria 29.38% 35.74% 33.42% Alphaproteobacteria 12.81 % 9.40 % 15.99 % Betaproteobacteria 6.22 % 7.50 % 3.38 % Deltaproteobacteria 0.81 % 2.68 % 1.15 % Gammaproteobacteria 10.12 % 16.92 % 13.43 % Bacteria Spirochaetes 0.00% 0.01% 0.00% Bacteria Verrucomicrobia 1.20% 3.37% 1.12% Bacteria Other 0.47% 0.93% 1.32%
45 5 DISCUSSION
In the following chapter the results obtained and displayed in chapter 4 will be discussed to accept or reject my hypotheses which in a short repetition are:
1) Thawing of high Arctic permafrost soil will lead to increased microbial activity. 2) High microbial activity at the beginning of the incubation experiment due to the increase availability of nutrients, which declines over time due to nutrients becoming limited. 3) Low content of organic material in the active layer due to the lack of vegetation. 4) Higher diversity and abundance within the upper soil layers and decreasing with depth. 5) Microbial activity decreases with soil depth as a result of expected lower diversity in deeper soil layers as well as decreasing nutrients concentration with depth.
5.1 Statistical errors
The applications of F-test and Student‟s t-test on the soil properties to test for significant difference were performed on non-standardized data. This means that there is a chance of making type I and II errors. A type I error is when a false positive is acquired (H0 is rejected), while a type II error is when a false negative is acquired (H0 is not rejected), but in reality the opposite is true. The level of significance is used to either reject or accept H0 and to state if a significant difference is obtained between data. The lower the level of significance, the higher is the risk of making one of these errors (Fowler et al. 1998).
No significant difference was obtained in pH throughout the soil core, but between the YP and OP in the incubated soil. By looking at figure 12 it can be seen that there is a clear decrease in pH with depth before incubation, whereas no difference can be seen between the YP and OP after incubation. This suggests that both type I and type II errors occurred here.
- NO3 concentration did obviously change in several depths during incubation (figure 14), yet only four were significant. This suggests that type II error occurred here as well.
Taken together, all significant difference should be interpreted with some skepticism.
5.2 Soil properties
5.2.1 Soil temperature, moisture & conductivity indicate depth of active layer
By definition, permafrost soils exist at temperatures below 0 °C. During August, the temperature at 95 cm depth did come close to 0 °C, but never above (see figure 9). Based on the temperature data I
46 assume that the permafrost layer begins close to 95 cm depth. Due to the interval gap between the measurement of 45 and 95 cm is it not possible to narrow it further down.
An elevated presence of salts was observed at 60-70 cm depth and increased soil moisture at 90-100 cm (see figure 11). The increased soil moisture indicates water accumulation. The increased conductivity indicates deposition of salts, suggesting water had been trapped at that depth probably the year before the soil core was sampled. The water then migrated upon thawing or disappeared due to high evaporation (French 2007). This suggests that the transient layer was about 60-70 cm depth in the previous year whereas it was between 70 and 90 cm the year of the sampling and the permafrost layer begins below.
5.2.2 pH
The change in pH during incubation is caused by microbial activity as described by Heynes (1986). PH did not change in the top soil (0-20 cm) which correlates to the low activity rate. Throughout the soil core pH changed mostly from 40 cm to 200 cm depth which also correlates to the increased CO2 production (figure 16). The largest decrease in pH was around the transition layer at 80-90 cm depth (figure 12).
Under anaerobic conditions it is likely that fermentation occurs. The fermentation of carbon by microorganisms is an anaerobic process that takes place much slower than oxidative respiration and results in the production of short-chained fatty acids as well as CO2 and CH4 which lower pH (Brown et al. 2010). The large decrease in pH at 80-90 cm can be explained by the production of short-chained fatty acids. Together with the increased soil moisture and CH4 emission it appears that the soil atmosphere in those samples was anaerobic and fermentation occurred.
- + 5.2.3 DOC, NO3 & NH4
+ Significantly higher concentrations of NH4 were obtained in the permafrost compared to the AL but - + no increase in NO3 . This corresponds to the high NH4 concentrations detected in permafrost melt water by Elberling et al. (2010), suggesting that N2O can be produced immediately upon thawing.
+ NH4 can be used in two ways: (i) through nitrification being transformed into nitrate and (ii) + assimilation where NH4 is taken up by microorganisms (Šimek & Cooper 2002). Because I did not - detect an increase in NO3 concentration after incubation, I assume that assimilation occurred, resulting + in the decrease of NH4 in the soil after incubation and the negligible N2O production rated indicate - that NO3 was not transformed into N2O or N2.
47 - The high concentration of NO3 in the AL is in relation to the soils conductivity and moisture. At the depths 0-10 cm and 60-70 m, the concentrations did not decrease in the incubated soil which - corresponds to the conductivity data. This suggests that NO3 was deposited at those depths, which is a - rare occurrence in the arctic. NO3 is a negative charged ion that does not bind to negatively charged soil particles and is therefore able to migrate with water down the core.
- The change in NO3 concentration at 90-100 cm depth is in relation to the increased soil moisture detected at the same depth. This suggests an anaerobic soil atmosphere and the opportunity for denitrification to occur.
It has to be kept in mind that the measured concentrations are representing a snapshot referring to the moment the samples were extracted. Of course the measured concentrations depend on the production and consumption rate by microbes which I did not measure. These may be estimated using 15N pool dilution techniques. It is assessed that the production and consumption will quickly increase when the temperature will rise thereby stimulating growth and activity.
DOC represents a small fraction of the soil organic material and depends on the production and consumption by microorganisms. It is a food supplement, supporting microbial growth (Kirchman et al. 1991). Low concentrations indicate either small amount of available organic C, therefore little growth, or effective decomposition of all DOC. Changes in DOC concentration suggests microbial activity. I observed significantly higher DOC concentrations in the permafrost compared to the AL. This supports the expectation that warming will result in the release of old, sequestrated carbon (Zimov et al. 2009), thereby enhancing microbial growth and activity and correlates to the other studies (Schmidt et al. 2011, Wild et al. 2014).
5.2.4 Total C, total N & total P
Soils in desert areas are known to contain less than 0.5 % organic C, but I detected around 2 % which corresponds to the organic matter content in agricultural soils (Griffin et al. 2013). This suggests that the method used did not measure the amount of total organic C, but the amount of total C including inorganic compounds such as carbonates and minerals. The amount of total organic C could be used to estimate the potential CO2 production in the soil, but not the amount of total C. Therefore I ignore total C as an influencing factor of microbial activity and focus on total N and total P.
As it can be seen on figure10, the increased concentration of total N in the YP suggest increased nutrients availability which stimulates microbial decomposition in that soil layer. As described by
Elberling et al. (2010), this nutrient pool supports N2O production for a short temporal burst, but longer-term N2O production requires external N input. Total P seems to have no correlation to microbial activity.
48 All together, the measured data show low organic content in the permafrost-affected soil as described by Campbell et al. (1998), Wagner (2008) and Masson-Delmotte et al. (2012) and therefore I can accept my third hypothesis.
5.3 Microbial activity
5.3.1 CO2 flux data
Since no vegetation was present in the samples, CO2 production observed during laboratory incubations represent only soil microbial respiration (Brummel & Siciliano 2011). The little quantity + of respiration in the AL is to be explained by the lack of nutrients, e.g. NH4 . At 90 cm depth an increase in the CO2 flux was observed, which stems from the increased nutrient availability upon thawing as shown by Kvenvolden & Lorensen (1993), Schuur et al. (2009) and Elberling et al. (2013).
The CO2 flux data show highest production in the YP and the upper OP (100-260 cm depth). By comparing these data to the amount of 16S rRNA genes present, with highest representation in the OP (180-300 cm depth), no pattern can be seen (see figure 1A in Appendix 2). It seems that most bacteria in the YP and the upper OP were active. However, this is not supported by the amount of total N. Total N is highest in the YP, but low in OP, therefore limiting microbial activity in OP. Thus I conclude that a lot of non-viable cells are present and only a few of the high quantity of bacterial genes are active.
The negative fluxes indicate CO2 consumption rather than production. Consumption can be the result of water capturing CO2. It is classified as an acid gas, easily dissolved in water and produces hydrogencarbonates (also called bicarbonates) when in contact with water. Bicarbonates (HCO-) are an intermediate form in the deprotonation of carbonic acid (H2CO3), thereby lowering the soils pH (Lal et al. 2003). This can explain the negative fluxes for each sampling period, but not for the overall flux. Based on soil moisture, I assume eventually frozen water to thaw during the incubation. The liquid water then places itself on top of the soil as a thin layer, capturing and dissolving CO2 when trying to diffuse to the atmosphere. However, moisture in the AL is very low and the pH change is minimal.
Therefore it seems unlikely that CO2 was dissolved in water.
Cyanobacteria were present in the soil core, with highest abundance in the AL (see table 1). Yet the water content remains low in the AL and the soil was incubated in the dark with exposure to light only during gas sampling periods. Therefore photosynthesis seems unlikely to occur and cannot explain the
CO2 consumption. On the other hand, chemolithoautotrophs can reduce CO2 to make organic compounds for biosynthesis or create a store of chemical energy through the Calvin Cycle using the key enzyme ribulose 1,5-biosposphate caroxylase (RuBisCO) (Swingley et al. 2009, Hügler & Sievert 2011). Especially the surface soil is exposed to higher climate fluctuations due to seasonality. This
49 increases stress for soil bacteria and the need for energy storage. Proteobacteria and Nitrospirae are known for application of chemosynthesis (Rae et al. 2013). The great abundance of Proteobacteria within the AL and the negative CO2 fluxes suggest that CO2 was actively fixed by these organisms. To test this theory the expression of RuBisCO genes should be investigated.
5.3.2 Statistical correlation between CO2 flux data & soil properties
The Spearman‟s rank correlation indicates that there was only a significant correlation between CO2 + flux and NH4 concentration, total N, total P, conductivity and soil moisture throughout the soil core.
This does support my first hypothesis that CO2 production will be affected by increased availability of nutrients as shown in other studies as mentioned previously.
When looking at the different layers individually, different soil properties affect CO2 production. In the AL, CO2 production was affected by DOC concentration, total N, conductivity and water content. - In the YP a correlation between CO2 and NO3 concentration, pH, total N and conductivity was detected. In the OP there is only a correlation between CO2 production and conductivity. This change in correlation indicates that with increasing depth, less soil properties have an actual affect on the CO2 production. The only property that had a correlation throughout all three layers was conductivity.
Conductivity is a measure of the capability to pass electrical flow and is greatly influences by the total concentration of salts present (salinity). The major soluble salts in soils are the cations sodium (Na+), 2+ 2+ + - 2- calcium (Ca ), magnesium (Mg ) and potassium (K ) and the anions chloride (Cl ), sulfate (SO4 ), - 2- - bicarbonate (HCO3 ), carbonate (CO3 ) and nitrate (NO3 ) (Shi & Wang 2005). Some of these ions are better to pass electrical flow than others. Therefore conductivity is not a measurement of the concentration of ions present. But it is safe to say that the more ions present, the higher the conductivity.
Salinity affects microorganisms by reducing microbial biomass and activity under high salt concentrations due to osmotic stress and toxic ions resulting in cell drying and lysis (Yan et al. 2015). Microorganisms have the ability to adapt or tolerate such stress by accumulating osmolytes (Sagot et al. 2010). The observed increase of CO2 production and conductivity with depth suggests that the bacterial community is well adapted to cope with osmotic stress.
5.3.3 CH4 flux data
Methanogenesis occurs under anaerobic conditions and the presence of methanogenic archaea. The lack of an anaerobic atmosphere as well as little representation of methanogenic archaea (0.08 %) can explain the low CH4 flux. But as to be seen on figure 16, the highest CH4 emission was in the
50 transition zone between the AL and the YP. At the same depth, the water content was highest (figure 10), indicating a rather anaerobic atmosphere.
CH4 can emit from soil in three ways: (i) through plant roots, (ii) through air bubbles, and (iii) through diffusion (Gray et al 2014). Option (i) and (ii) are unlikely since no vegetation was found at the field site and the soil wasn‟t wet enough to result in the construction of air bubbles. CH4 can thus be produced in anaerobic layers and diffuse up. When reaching the aerobic layer, bacterial methanotrophs belonging to the class‟s gamma- and betaproteobacteria oxidize CH4 to CO2 and H2O (Hanson & Hanson 1996, Thauer et al. 2008, Gray et al. 2014). I am not able to determine if this was infect true, since I didn‟t incubate the soil core as a whole, but in discrete intervals. However, it seems likely that the produced CH4 in the deeper soil layers would not reach the atmosphere due to the greater presence of gamma- and betaproteobacteria found throughout the soil core. Oxidation of CH4 does also explain the negative flux observed in the monthly sampling.
Lack of production under anaerobic incubation is explained by limited nutrient availability as well as the low presence of methanogenic arachaea.
5.3.4 N2O flux data
N2O emission from soils is linked to soil moisture, oxygen availability and amount of labile nitrogen and carbon (Mørkved et al. 2007, Elberling et al. 2013). The lack of microbial activity explains the lack of N2O production, although the low rates of available water and nitrogen can explain the low
N2O production observed.
N2O production was mainly seen in the OP, whereas consumption was observed within the AL and YP. Denitrification occurs only under anaerobic conditions and is generally recognized as a major mechanism contributing to N2O production and is the only known biological process for N2O consumption (Hu et al. 2015). N2O consumption appears to be exclusively due to the respiratory N2O reductase Nos as part of the biochemical pathway dissimilative nitrate reduction, which is the stepwise - - reduction of NO3 → NO2 → NO → N2O → N2 as the end product (Tiedje 1988). I only analyzed the production of N2O and not N2; therefore I assume that N2O was reduced to N2 which explains the negative flux both obtained in the aerobic and anaerobic incubation experiment.
Taken all of these results together, I can partially accept my first hypothesis. I did observe an increase in the production of CO2 under aerobic conditions, but nothing under anaerobic conditions, suggesting that soil respiration is the major metabolic pathway used by microorganisms in high Arctic permafrost soil. Due to the calculation of an overall flux and the exclusion of data obtained from each sample
51 period, I am not able to verify my second hypothesis. I did not observe a change in microbial activity due to nutrient limitation.
5.4 Microbial community
5.4.1 Archaeal diversity
As expected, the bacterial diversity was higher than the diversity of archaea and fungi. The diversity of archaea was minimal whereas no fungi were present at all. Surprisingly, I did find a greater abundance of the Crearchaeota with greatest abundance of the genus Nitrososphaera, an anaerobe ammonia- oxidizing archaea (anammox) rather than the expected Euryarchaeotea. The very low amount of methanogenic archaea explains the low CH4 production, as described in the previous section 5.3.3
(CH4 flux data), and correlated well with the low soil moisture (Waldrop et al. 2010).
5.4.2 Bacterial diversity
The high significant level in diversity obtained from the ANISMO indicates that the bacterial community is highly diverse between the different soil layers. Figure 19 shows groupings of sequences obtained from the different layers. The position of the bubbles indicate the distance between the number of sequences; the greater the distance, the more diverse the communities are at the different depths. The measured figure suggests the diversity increases with depth, which is in contradiction to my expectations. However, it is important to remember that the numbers of sequences were standardized and figure 19 is only an illustration of the relation between each sample and not an actual representation. Also, due to the constant subzero temperatures in permafrost, dead or compromised microbial cells may remain well preserved and contribute to total microbial counts (Rivkina et al. 2004, Willerslev et al. 2004).
The isolated phyla correspond to phyla found in permafrost samples from other studies (D‟Almico et al. 2006, Steven et al. 2007, Johnson et al. 2007, Yergeau et al. 2010, Hinsa-Leasure & Bakermans 2013). Proteobacteria make up the largest bacterial phylum and are all gram-negative (table 1), addressing a wide diversity of energy-generating mechanisms (Madigan et al. 2012). Their predominance indicates their resilience for cold survival.
Actinobacteria are known to adapt to stress by entering a dormant, but viable state. Their high GC content may help to reduce DNA damage owing to cold temperature and high salt concentrations (Jansson & Taş 2014). The subclass Rubrobacteraceae, which was detected throughout the soil core, is known to be radiation and desiccation tolerant which are beneficial for survival in permafrost (Wilhelm et al. 2012).
52 Firmicutes have a low GC content in contrast to Actinobacteria, but are able to produce endospores which are resistant to desiccation and can survive extreme conditions (Steven et al. 2008). Chloroflexi are ubiquitous in permafrost soils, but many functions remain undiscovered. Bacteroidetes are gram- negative, non-sporeforming and widely distributed in the environment (Taş et al. 2014).
Together with the qPCR data, showing an increase of bacterial 16S rRNA genes with depth (figure 18), these results indicate that bacterial diversity and abundance is lowest in the upper soil layer and increases with depth. This does not support my fourth hypothesis, which I therefore have to reject.
Figure 19: Visualization of the correlation between bacterial diversity and CO2 production in the different soil layers. Blue bubbles represent the AL, read bubble represent the YP and green bubbles represent the OP. The position of the bubbles is random whereas their size correlates to the amount of CO2 produced at that depth as ng CO2 produced per g dry soil per day
(left box); the greater the size of the bubble, the greater CO2 production.
5.4.3 Community composition affected by soil properties
As already mentioned, the bacterial community was significantly diverse. This vertical variation is influenced by soil properties that changed with soil depth, such as pH, total N and water availability (Kim et al. 2014). Soil pH is considered the most important factor influencing microbial community structure (Campbell et al. 2010, Chu et al. 2010), which again can be related to moisture availability (Rousk et al. 2010).
Figure 19 illustrates the relationship between CO2 production at the different depths and the bacterial community composition. An overall tendency for increased CO2 production with increasing depth can be seen. This type of plot was also done for total P, total N, conductivity, soil moisture and the
53 + - concentrations of DOC, NH4 and NO3 in the non-incubated soil (data not shown due to lack of + correlation; but both conductivity and NH4 concentration showed a tendency to increase with soil - depth, whereas NO3 concentration decreases with depth). All plots had a 2D stress value of 0.31. According to Clarke et al. (2014), a stress value >0.3 indicates that the points are close to being arbitrarily placed. Consequently these data are assessed as less reliable and lead me to the conclusion that any correlation between the soil properties and bacterial diversity is random. Therefore I can reject my fifth and last hypothesis.
54 6 CONCLUSION
The objective of this thesis was to investigate how microbial activity in a high Arctic permafrost core will change through increased temperatures under aerobic and anaerobic conditions. Nothing is to conclude from the anaerobic incubation experiment.
Under the aerobic incubation experiment, increased activity was observed in the form of elevated CO2 production at most depths over the full duration of the experiment, while CH4 and N2O production was very low. The soil core had an overall low organic content limiting microbial activity, even though statistically analyses indicated CO2 production was influenced by some soil properties. Production was mostly influenced in the AL and with depth the amount of properties affecting CO2 production decreased.
The microbial community was clearly dominated by bacteria, with very low representation of archaea and no fungi. Bacterial diversity and abundance was low in the AL and increased with depth. The community composition was significantly diverse throughout the soil core, especially between the AL and OP.
All together I can conclude that increased temperatures do influence the microbial activity in high Arctic permafrost. It is important to note that this thesis only focuses on the community of a single soil core and does not reflect any comprehensive survey of permafrost environments.
55 7 PERSPECTIVES
Due to the limited timeframe and resources, all of the following recommendations could not be done as part of this thesis, but are of interest within the topic.
For future work I recommend that samples from several soil cores should be used. The work on one core alone is not representative, whereas replicates will give a better insight into permafrost environments.
The work on amplified 16S rRNA genes from DNA isolates gave an estimate on how many and which organisms were present, but includes both viable and non-viable cells. To exclude the dead ones, qPCR and sequencing should have been done on RNA isolates as well to. Also, by amplifying different genes involved in different processes such as nitrification, Calvin Cycle and methane oxidation a better image is obtained on the function of microbes in these environments and how they respond to climate change.
In this thesis I focused on the changes in microbial activity as a consequence of warming. Another aspect would be to test for changes in activity as a consequence of factors other than temperature. One example is the simulation of increased rain in the top soil. The set up would be like describes for the incubation experiments by filling serum bottles with homogenized soil, incubation and measurement of produced gas with the addition of water. For this the soils water holding capacity needs to be known. Soil should be placed in a funnel containing a thin glass filter at the bottom, filled with water and left over night at 4 °C. The soil is then weighted, burned and weighted again. The weight difference estimates the amount of lost fluids and shows how much water can be absorbed by the soil. This amount is then added to the soil before incubation. Another experiment would be to investigate how activity changes when nutrients would not become limited. To do so, substrates containing carbon, nitrogen and phosphorous should be added. For this I would have at least 7 setups, each containing different substrates, by adding the substrates individually, in combinations of two and in combination of all three. Eventually more setups could be made, with different substrate concentrations. This experiment would give an estimate on how big the potential for activity can be.
56 8 ACKNOWLEDGEMENTS
First of all I want to thank Anders Priemé and Morten Schostag for academic supervision during this thesis, showing me how to work scientifically on a professional level and all their help in the lab and feedback on written assignments. I am grateful to Bo Elberling (CENPERM) for providing the samples used to work on for this thesis.
My gratitude goes to Samuel Faucherre (CENPERM) for helping me with the setup for the incubation experiment, Nanna Slaikjer Petersen for helping me with the Illumina MiSeq protocol, Luma George Odish and Martin Steen Mortensen (UCPH) for sequencing my samples as well as Mads Bolander Jensen (UCPH) for the bioinformatical analyses of the sequencing raw data.
A specially thanks goes to all laboratory technicians at the University of Copenhagen, Gosha Sylvester, Annette Spangenberg and Esben Nielsen from the Section for Terrestrial Ecology, Anette Hørdum Løth from the Section for Microbiology and Maja Holm Wahlgren from CENPERM as well as the laboratory technicians at the Geological Survey of Denmark and Greenland Pia Bach Jackobsen, Pernille Stockmarr and Spire Maja Kiersgaard for helping me with the practical laboratory work in addition to analyzing my data.
I want to thank Laura Rasmussen for helping me with the geophysical aspect in this thesis as well as Merian Haugwitz (DTU) and Jana Vorískova (Lawrence Berkeley National Laboratory) for feedback regarding fungal analyses.
Last but not least, an extraordinary thanks to Sven Helbig for statistical support, Morten Rasmussen for technical support and Stephan Helbig for constructive feedback on the written assignments.
57 9 REFERENCES
Aanderud, Z.T., Jones, S.E., Schoolmaster Jr., D.R., Fierer, N., and Lennon, J.T., 2013, Sensitivity of soil respiration and microbial communities to altered snowfall, Soil Biolo Biochem, 57(11):217- 227.
ACIA, 2004, Arctic Climate Impact Assessment, Cambridge University Press, Cambridge.
Allaby, M., 2015, A Dictionary of Geology and Earth Sciences, Oxford University Press, Cambridge, 4th edition, pp. 672.
AMAP, 2011, Snow, Water, Ice and Permafrost in the Arctic (SWIPA): Climate Change and the Cryosphere, Arctic Monitoring and Assessment Programme (AMAP), Oslo, pp. 538.
Anisimov, O.A., 2007, Potential feedback of thawing permafrost to the global climate system through methane emission, Environ Res Lett, 2:045016.
Bakermans, C., Tsapin, A.I., Souza-Egipsy, Gilinchinsky, D., and Nealson, K.H., 2003, Reproduction and metabolism at -10 °C of bacteria isolated from Siberian permafrost, Environ Microbiol, 5(4):321-326.
Bardgett, R.D., 2005. The biology of soil: a community and ecosystem approach, 1st editiontio, Oxford University Press, pp. 254
Blattner, F.R., Plunkett, G., Bloch, C.A., Perna, N.T., Burland, V., Riley, M., Collado-Vides, J., Glasner, J.D., Rode, C.K., Mayhew, G.F., Gregor, J., Davis, N.W., Kirkpatrick, H.A., Goeden, M.A., Rose, D.J., Mau, B., and Shao, Y., 1997. The Complete Genome Sequence of Escherichia coli K-12, Science, 277:1453-1462.
Bockheim, J., and Tarnocai, C., 1998, Recognition of cryoturbation for classifying permafrost-affected soils. Geoderma, 81:281-293.
Brook, E.J., Sowers, T., and Orchardo, J., 1996, Rapid variations in atmospheric methane concentration during the past 110,000 years, Science, 273:1087-1091.
Burn, C., 2012, Permafrost Distribution and Stability, in French, H., and Slaymaker, O., (eds.) Changing Cold Environments: A Canadian Perspective, Wiley-Blackwell, 1st edition, pp. 126-146.
Brummell, M.E., and Siciliano, S.D., 2011, Measurement of Carbon Dioxide, Methane, Nitrous oxide, and Water Potential in Soil Ecosystems, in Klotz, M.G., and Stein, L.Y., (eds.) Methods in Enzymology, V.496, pp. 115-37.
58 Brown, J., Ferrians, O.J.J., Heginbottom, J.A., and Melnikov, E.S., 1997, Circum-Arctic Map of Permafrost and Gound Ice Conditions, Scale 1:10.000.000, U.S. Geol Surv.
Brown, T.L., LeMay, H.E., Bursten, B.E., Murphy, C.J., Langford, S.J., and Sagatys, D., 2010, Chemistry: The central science: a broad perspective, Pearson, pp. 1190.
Campbell, I.B., Claardige, G.G.C., Campbell, D.I., and Balks, M.R., 1998, The Soil Environment of the Mcmurdo Dry Valleys, Antarctica, in Priscu, J.C., (ed.) Ecosystem Dynamics in a Polar Desert: the Mcmurdo Dry Valleys, Antarctica, Amer Geophys Union, pp. 297-322.
Campbell, B.J., Polson, S.W., Hanson, T.E., Mack, M.C., and Schuur, E.A.G., 2010, The effect of nutrient deposition on bacterial communities in Arctic tundra soil, Environ Microbiol, 12:1842- 1854.
The Carbon Dioxide Information Analysis Center (CDIAC), 2011, Atmospheric lifetime of different gases, ChartsBin statistics collector team 2011; http://chartsbin.com/view/2407 - website visited on November 24th, 2016
Centre d‟études nordiques, http://www.cen.ulaval.ca/bylot/ecomon-carbone-mares.htm - website visited on December 1st, 2016
Chapman, S.J., 2010, Carbon Capture: Sequestration and Storage, in Hester, R.E., and Harrison, R.M., (eds.) Issues in Environmental Science and Technology, Royal Society of Chemistry, V. 29, pp. 179-202
Chen, G., Zhu, H., and Zhang, Y., 2003, Soil microbial activites and carbon and nitrogen fixation, Res Microbiol., 154(6):393-398.
Christensen, T.R., Johansson, T.R., Akerman, H.J., Mastepanov, M., Malmer, N., Friborg, T., Crill, P., and Svensson, B.H., 2004, Thawing sub-arctic permafrost: Effects on vegetation and methane emissions, Geophy Res Lett, 31(4):L04501.
Chu, H., Fierer, N., Christian, L.L., Caporaso, J.G., Knight, R., and Grogan, R., 2010, Soil bacterial diversitz in the Arctic is not fundamentally different from that foun in other biomes, Environ Microbiol, 12:2998-3006.
Chu, H., Neufeld, J.D., Walker, V.K. and Grogan, P., 2011, The influence of vegetation type on the dominant soil bacteria, archaea, and fungi in a low arctic tundra landscape, Soil Biol Biochem, 75:1756-1765.
Clarke, K.R., Gorley, R.N., Somerfield, P.J., and Warwick, R.M., 2014, Change in Marine Communities: An Approach to Statistical Analysis and Interpretation, Primer-E Ltd, 3rd edition, pp. 262.
59 Clemmensen, K.E., Bahr, a, Ovaskainen, O., Dahlberg, A., Ekblad, A., Wallander, H., Stenlid, J., Finlay, R.D., Wardle, D.A., Lindahl, B.D., 2013, Roots and associated fungi drive long-term carbon sequestration in boreal forest, Science, 339:1615-1618.
Cockell, C.S., Rehberg, P., Horneck, G., Wynn-Williams, D.D., Scherer, K. and Gugg-Helminger, A., 2002, Influence of ice and snow covers on the UV exposure of terrestrial microbial communities: dosimetric studies. J Photochem Photobiol, 68:23-32.
Conant, R.T., Ryan, M.G., Ågren, G.I., Birge, H.E., Davidson, E.A., Eliasson, P.E., Evans, S.E., Frey, S.D., Giardina, C.P., Hopkins, F.M., Hyvönen, R., Kirschbaum, M.U.F., Lavallee, J.M., Leifeld, J., Parton, W.J., Steinweg, J.M., Wallenstein, M.D., Wetterstedt, J.Å.M., Bradford, M.A., 2011, Temperature and soil organic matter decomposition rates – synthesis of current knowledge and a way forward, Glob Chang Biol, 17:3392–3404.
Crawford, R.L., and Newcombe, D.A., 2008, The Potential for Extant Life in the Soils of Mars, in Dion, P., and Nautiyal, C.S., (eds.) Microbiology of Extreme Soils, Springer-Verlag Berlin Heidelberg, pp. 225-243.
Crawford, R.M.M., 2008, Plants at the Margin – Ecological Limits and Climate Change, Cambridge University Press, pp. 478.
Crutin, D., Campbell, C.A., and Jalil, A., 1997, Effects of acidity on mineralization: pH-dependence of organic matter mineralization in weakly acidic soils, Soil Bio Biochem, 30(1):57-64.
Daanen, R.P., Ingeman-Nielsen, T., Marchenko, S.S., Romanovsky, V.E., Foged, N., Stendel, M., Christensen, J.H., Hornbech Svendsen, K., 2011, Permafrost degradation risk zone assessment using simulation models, Cryosph, 5:1043-1056.
D‟Almico, S., Claverie, P., Collins, T., Georlette, D., Gratia, E., Hoyoux, A., Meuwis, M.-A., Feller, G., and Gerday, C., 2002, Molecular basis of cold adaptation, Phil Trans R Soc B, 357:917-925.
D‟Almico, S., Collins, T., Marx, J.-C., Feller, G., Gerday, C., and Gerday, C., 2006, Psychrolphilic microorganisms: challenges for life, EMBO reports, 7(4):374-455.
Davidson, E.A., Savage, K., Verchot, L.V., and Navarro, R., 2002, Minimizing artifacts and biases in chamber-based measurement of soil respiration, Agricultural Forest Meteorology, 113:21-37.
De Maayer, P., Anderson, D., Cary, C., and Cowan, D.A., 2014, Some like it cold: understanding the survival strategies of psychrophiles, EMBO Rep, 15(5):508-517.
Deming, J.W., 2002, Psychrophiles and polar regions, Curr Opin Microbiol, 5:301-309.
60 Deslippe, J.R., Hartmann, M., Simard, S.W., Mohn, W.W., 2012, Long-term warming alters the composition of Arctic soil microbial communities, FEMS Microbiol Ecol, 82:303-315.
Dobinski, W., 2011, Permafrost, Earth Sci Rev, 108:158-169.
Elberling, B., Chrisitansen, H.H., and Hansen, B.U., 2010, High nitrous oxide production from thawing permafrost, Nature Geosci, 3:332-335.
Elberling, B., Michelsen, A., Schädel, C., Schuur, E.A.G., Christiansen, H.H., Berg, L, Tamstorf,
M.P., and Sigsgaard, C., 2013, Long-term CO2 production following permafrost thaw, Nature Clim Chang, 3:890-894.
Farbrot, H., Isaksen, K., Etzelmüller, B., and Gisnås, K., 2013, Ground Thermal Regime and Permafrost Distribution under a Changing Climate in Northern Norway, Permafrost Periglac Process, 24(1):20-38.
Feller, G. and Gerday, C., 2003, Psychrophilic enzymes: hot topics in cold adaptation, Nature Rev Microbiol, 1:200–208.
Fierer, N., Jackson, J.A., Vilglys, R., and Jackson, R.B., 2005, Assessment of Soil Microbial Community Structure by Use of Taxon Specific QuantitativePCR Assays, Appl Environ Microbiol, 71(7):4117-4120.
Fierer, N., and Jackson, R.B., 2006, The diversity and biogeography of soil bacterial communities, Proc Natl Acad Sci USA, 103:626-631.
Fowler, J., Cohen, L., and Jarvis, P., 1998, Practical Statistics for Field Biology, Wiley, 2nd edition, pp. 259.
French, H., and Shur, Y., 2010, The principles of cryostratigraphy, Earth Sci Rev, 101:190-206.
French, H.M., 2007, The periglacial environment, Wiley, 3rd edition, pp. 478.
Friedmann, E.I., 1994, Permafrost as microbial habitiat, in Gilichinsky, D., (ed.) Viable Microorganisms in Permafrost, Russ Acad Sci, Pushchino, pp. 21-26
Gilichinsky, D., and Rivkina, E., 2011, Permafrost Microbiology, in Reitner, J., and Thiel, V., (eds.) Encyclopedia of Geobiology, Springer Netherlands, V.1, pp. 927.
Graham, D.E., Wallenstein, M.D., Vishnivetskaya, T.A., Waldrop, M.P., Phelps, T.J., Pfiffner, S.M., Onstott, T.C., Whyte, L.G., Rivkina, E.M., Gilichinsky, D.A., Elias, D.A., Mackelprang, R., VerBerkmoes, N.C., Hettich, R.L., Wagner, D., Wullschleger, S.D., Jansson, J.K., 2011, Microbes in thawing permafrost: the unknown variable in the climate change equation, ISME J, 6:709-712.
61 Gray, N.D., McCann, M.C., Christgen, B., Ahammad, S.Z., Roberts, J.A., and Graham, D.W., 2014, Soil geochemistry confines microbial abundances across an arctic landscape; implications for net carbon exchange with the atmosphere, Biogeochem, 120:307-317.
Gregorich, E.G., Hopkins, D.W., Elberling, B., Sparrow, A.D., Novis, P., Greenfield, L.G., and
Rochette, P., 2006, Emission of CO2, CH4 and N2O from lakeshore soils in an Antarctic dry valley, Soil Biol Biochem, 38:3120-3129.
Griffin, E., Hoyle, F.C., and Muryphy, D.V., 2013, Soil organic carbon, in Report card on sustainable natural resource use in Agriculture, Department of Agriculture and Food, Western Australian Agriculture Authority, pp. 16.
Guo, L., Ping, C.L., and Macdonals, R.W., 2007, Mobilization pathways of organic carbon from permafrost to Arctic rivers in a changing climate, Geophys Res Lett, 34(13):L13603.
Hanson, R.S., and Hanson, T.E., 1996, Methanotrophic bacteria, Microbiol Rev, 60(2):439-471.
Harris, C., Arenson, L.U., Christiansen, H.H., Etzelmüller, B., Frauenfelder, R., Gruber, S., Haeberli, W., Hauck, C., Hölzle, M., Humlum, O., Isaksen, K., Kääb, A., Kern-Lütschg, M.A., Lehning, M., Matsuoka, N., Murtin, J.B., Nötzli, J., Phillips, M., Ross, N., Seppälä, M., Springman, S.M., and Von der Mühll, D., 2008, Permafrost and climate in Europe: Monitoring and modeling thermal, geomorphologcial and geotechnical responses, Earth Sci Rev, 92:117-171.
Haynes, R.J., 1986, Mineral Nitrogen in the Plant-Soil System, Academic Press, Orlando, pp. 483.
Heikoop, J.M., Throckmorton, H.M., Newman, B.D., Perkins, G.B., Iversen, C.M., Chowdhury, T.R., Romanovsky, V., Graham, D.W., Norby, R.J., Wilson, C.J., and Wullschleger, S.D., 2015, Isotopic identification of soil and permfrost nitrate sources in an Arctic tundra ecosystem, J Geophys Res Biogeosci, 120:1000-1017.
Heimann, M., and Reichstein, M., 2008, Terrestrial ecosystem carbon dynamics and climate feedbacks, Nature, 451:289-292.
Herbert, E.R., Boon, P., Burgin, A.J., Neubauer, S.C., Franklin, R.B., Ardón, M., Hoppensperger, K.N., Lamers, L.P.M., and Gell, P., 2015, A global perspective on wetland salinization: ecological consequences of a growing threat to freshwater wetlands, Ecosphere, 6(10):1-4.
Hinkel, K.M., and Nelsen, F.E., 2003, Spatial and temporal patterns of active layer thickness at Circumpolar Active Layer Monitoring (CALM) sites in northern Alaska, 1995–2000. Geophys Res, 108:9-13.
Hinsa-Leasure, S., and Bakermans, C., 2013, Diversity of Bacteria in Permafrost, in Yumoto, I., (ed.) Cold-adapted Microorganisms, Caister Academic Press, pp. 1-12.
62 Hollesen, J., Matthiesen, H., Møller, A.B., and Elberling, B., 2011, Permafrost thawing in organic Arctic soils accelerated by ground heat production, Nature Clim Chang, 5:574-578.
Hu, J., Inglett, K.S., Clark, M.W., Inglett, P.W., and Reddy, K.R., 2015, Nitrous oxide production and consumption by denitrification in grassland: Effects of grazing and hydrology, Sci Total Environ, 532:702-710.
Hügler, M., and Sievert, S.M., 2011, Beyond the Calvin Cycle: Autotrophic Carbon Fixation in the Ocean, Annual Rev Mari Sci, 3:261-289.
Ihrmark, K., Bödeker, I.T., Cruz-Martinez, K., Friberg, H., Kubartova, A., Schenck, J., Strid, Y., Stenlid, J., Brandström-Durling, M., Clemmensen, K.E., Lindahl, B.D., 2012, New primers to aomplify the fungal ITS2 region – evaluation by 454-sequencingof artificial and natural communities, FEMS Microbiol Ecol, 82(3):666-677.
Illeris, L., Michelsen, A., Jonasson, S., 2003, Soil plus root respiration and microbial biomass following water, nitrogen, and phosphorous application at a high arctic semi desert, Biochem, 65:15-29.
IPCC, 2007, Intergovernmental Panel on Climate Change, Synthesis Report. Cambridge University Press, Cambridge.
IPCC, 2013, Intergovernmental Panel on Climate Change, The Physical Science Basis. Cambridge University Press, Cambridge.
Jansson, J.K., and Taş, N., 2014, Thie microbial ecology of permafrost, Nature Rev, 12:414-425.
Johnson, J.M.F., Reicosky, D.C., Allmara, R.R., Sauer, T.J., Venterea, R.T., and Dell, C.J., 2005, Greenhouse gas contributions and mitigation potential of agriculture in the central USA, Soil Till Res, 83:73-94.
Johnson, S.S., Hebsgaard, M.B., Chistensen, T.R., Mastepanov, M., Nielsen, R., Munch, K., Brand, T., Gilbert, M.T.P., Zuber, M.T., Bunce, M., Rønn, R., Gilichinsky, D., Froese, D., Willerslev, E., 2007, Ancient Bacteria Show Evidence of DNA Repair, Proc Natl Acad Sci USA, 104:14401- 14405.
Jonasson S., Chapin, F.S.I. & Shaver, G.R., 2001, Biogeochemistry in the Arctic: Patterns, Processes, and Controls, in Schulze, E.-D., Harrison, S.P., Heimann, M., Holland, E.A., Lloyd, J.J., Prentice, I.C., and Schimel, D., (eds.) Global Biogeochenzical Cycles in the Climate System, Academic Press, New York, pp. 139-150
Jónsdóttir, I.S., 2005, Terrestrial Ecosystems on Svalbard: Heterogeneity, Complexity and Fragility from an Arctic Island Perspective, Biology and Environment, 105: 155-165.
63 Kim, H.Y., Jung, J.Y., Yergeau, E., Hwang, C.Y., Hinzman, L., Nam, S., Hong, S.G., Kim, O.-S., Chun, J., and Lee, Y.K., 2014, Bacterial community structure and soil properties of a subarctic tundra soil in Council, Alaska, FEMS Microbiol Ecol, 89:465-475.
Kimble, J.M., 2004, Cryosols, permafrost affected soils, Springer-Verlag Berlin Heidelberg, pp. 726.
Kirchman, D.L., Suzuki, Y., Garside, C., and Ducklow, H.W., 1991, High turnover rates of dissolved organic carbon during a spring phytoplankton bloom, Nature, 352:612-614.
Knoblauch, C., Beer, C., Sosnin, A., Wagner, D., and Pfeiffer, E.-M., 2013, Predicting long-term carbon mineralization and trace gas production from thawing permafrost of North Siberia, Glob Chang Biol, 19:1160-1172.
Kokelj, S.V., and Burn, C.R., 2005, Geochemistry of the active layer and near-surface permafrost, Mackenzie delta region, Northwest Territories, Canada, Can J Earth Sci, 42:37-48.
Koven, C.D., Ringeval, B., Friedlingstein, P., Ciais, P., Cadule, P., Khvorostyanov, D., Krinner, G., and Tarnocai, C., 2011, Permafrost carbon-climate feedbacks accelerate global warming. PNAS, 108:14769-14774
Kushner, D., 1981, Extreme environments: Are there any limits to life?, in Ponnamperuna, C., (ed.) Comets and the origin of life, Reidel, New York, pp. 241-248.
Kvenvolden, K.A., and Lorenson, T.D., 1993, Methane in permafrost: Preliminary results from coring at Fairbanks, Alaska, Chemosphere, 26:609-616.
Lal, R., 1995, Global soil erosion by water and carbon dynamics, in: Lal, R., Kimball, J., Levine, E., and Stewart, B.A., (eds.) Soil and Global Change, CRC/Lewis Publishers, pp.131-142.
Lal, R., Follet, R.F., and Kimble, J.M., 2003, Achieving soil carbon sequestration in the United States: A challenge to the poly makers, Soil Sci, 168(2):827-845.
Larose, C., Dommerque, A., and Vogel, T.M., 2013, The Dynamic Arctic Snow Pack: An Unexplored Environment for Microbial Diversity and Activity, Biology, 2(1):317-330.
Lauber, C.L., Hamady, M., Knight, R., and Fierer, N., 2009, Pyrosequencing-based assessment of soil pH as a predictor of soil bacterial community structure at the continental scale, Appl Environ Microbiol, 75:5111-5120.
Laughlin, R.J., and Stevens, R.J., 2002, Evidence for Fungal Dominance of Denitrification and Codenitritifaction in a Grassland Soil, Am J Soil Sci Soc, 66:1540-1548.
Lawrence, D.M., and Slater, A.G., 2005, A project of severe near-surface permafrost degradation during the 21st century, Geophys Res Lett, 32(24):L24401.
64 Lewis, K., Epstein, S., Godoy, V.G., and Hong, S.-H., 2007, Intact DNA in ancient permafrost, Trends Microbiol, 16(3):92-94
Lipson, D.A., Monson, R.K., Schmidt, S.K., and Weintraub, M.N., 2009, The trade-off between growth rate and yield in microbial communities and the consequences for under-snow soil respiration in a high elevation coniferous forest, Biogeochem, 95:23-35.
Mackelprang, R., Waldrop, M.P., DeAngelis, K.M., David, M.M., Chavarria, K.L., Blazwicz, S.J., Rubin, E.M., and Jannson, J.K., 2011, Metagenomic analysis of permafrost microbial community reveals a rapid response to thaw, Nature, 480:368-371.
Madigan, M., Martinko, J., Stahl, D., and Clark, D., 2012, Biology of Microorganisms, Pearson, 13th edition, pp. 1149.
Mannisto, M.K., Tiirola, M. and Haggblom, M.M., 2007, Bacterial communities in Arctic fields of Finnish Lapland are stable but highly pH dependent, FEMS Microbiol Ecol, 59:452-465.
Martin, E., and Hine, R., (ed.), Dictionary of Biology, Oxford University Press, V.7, pp. 736.
Masson-Delmotte, V., Swingedouw, D., Landais, A., Seidenkrantz, M.-S., Gauthier, E., Bichet, V., Massa, C., Perren, B., Jomelli, V., Adalgeirsdottir, G., Hesselbjerg Christensen, J., Arneborg, J., Bhatt, U., Walker, D. a., Elberling, B., Gillet-Chaulet, F., Ritz, C., Gallée, H., van den Broeke, M., Fettweis, X., de Vernal, A., Vinther, B., 2012, Greenland climate change: from the past to the future. Wiley Interdiscip Rev Clim Chang, 3:427-449.
Mazur, P., 1980, Limits to life at low temperatures and at reduced water contents and water activities, Orig Life, 10:137-159.
Morita, R.Y., 1975, Psychrophilic bacteria, Bacteriol Rev 39:144-167.
Murton, J.B., 2009, Global Warming and Thermokarst, in Margesin, R., (ed.) Permafrost soils, Springer-Verlag Berlin Heidelberg, V.16, pp. 185-203.
Muyzer, G., De Waal, E.C., Uitterlinden, A.G., 1993, Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA, Appl Environ Microb 59:695–700.
Mørkved, P.T., Dörsch, P., and Bakken, L.R., 2007, The N2O product ratio of nitrification and its dependence on long-term changes in soil pH, Soil Biol Biochm, 39:2048-2057.
Nelissen, V., Saha, B.K., Ruysschaert, G., and Boeckx, P., 2014, Effect of different biochar and fertiliyer types on N2O and NO emissions, Soil Biol Biochem, 70:244-255.
Nelson, F.E., 2003, Geocrylogoy. (Un)frozen in time, Science, 299:1673-1675.
65 Ozerskaya, S., Kochkina, G., Ivanushkina, N., and Gilichinsky, D., 2009, Fungi in Permafrost, in Margesin, R., (ed.) Permafrost soils, Springer-Verlag Berlin Heidelberg, V.16 , pp. 85-95.
Öqvist, M.G., Sparrman, T., Klemedtsson, L., Harryson Drotz, S., Grip, H., Schleucher, J., and
Nilsson, M., 2009, Water availability control microbial temperature responses in frozen soil CO2 production, Glob Chang Biol, 15(11):2715-2722.
Panikov, N.S., and Sizova, M.V., 2007, Growth kinetics of microorganisms isolated from Alaskan soil and permafrost in solid media frozen down to -35°C, FEMS Microbiol Ecol, 59:500–512.
Panikov, N.S., 2009, Microbial Activity in Frozen Soils, in Margesin, R., (ed.) Permafrost soils, Springer-Verlag Berlin Heidelberg, V.16 , pp. 346.
Passarge, S., 1920, Die Grundlagen der Landsschaftskunde, Friedrichen & Company, Hamburg, pp. 588.
Paul, E.A., and Clark, F.E., 1996, Soil Microbiology and Biochemistry, Academic Press, San Diegeo, 2nd edition, pp. 353.
Pina, M., Bize, A., Forterre, P., and Prangishvili, D., 2011, The archaeaviruses, FEMS Microbiol Rev, 35:1035-1054.
Payette, S., Delwaide, A., Caccianiga, M., and Beauchemin, M., 2004, Accelerated thawing of subarctic peatland permafrost over the last 50 years, Geophys Res Lett, 31:L18208.
Price, P.B., 2007, Microbial life in glacial ice and implications for a cold origin of life. FEMS Microbiol Ecol, 59:217-231.
Quinn, P.K., Bates, T.S., Baum, E., Doubleday, N., Fiore, A.M., Flanner, M., Fridlind, A., Garrett, T.J., Koch, D., Menon, S., Shindell, D., Stohl, A., and Warren, S.G., Short-lived pollutants in the Arctic: their climate impact and possible mitigation strategies, Atmos Chem Phys, 8:1723-1735
Rae, B.J., Long, B.M., Badger, M.R., and Price, G.D., 2013, Functions, Compositions, and Evolution
of the Two Types of Carboxysomes: Polyhedral Microcompartments That Facilitate CO2 Fixation in Cyanobacteria and Some Proteobacteria, Microbiol Mol Biol Rev, 77(3):357-379.
Rivkina, E., Gilichinsky, D., Wagener, S., Tiedje, J., and McGrath, J., 1998, Biogeochemical activity of anaerobic microorganisms from buried permafrost sediments, Geomicrobiol, 15(3):187-193.
Rivkina, E., Friedmann, E.I., McKay, C.P., and Gilichinsky, D., 2000, Metabolic activity of permafrost bacteria below the freezing point, Appl Environ Microbiol, 66(8):3230-3233.
Rivkina, E., Laurinavichius, K.., McGrath, J., Tiedje, J., Scherbakova, V., and Gilichinsky, D., 2004, Microbial life in permafrost, Adv Space Res, 33:1215-1221.
66 Rivkina, E., Shcherbakova, V., Laurinavichius, K.., Petrovskaya, L., Krivushin, K., Kraev, G., Pecheritsina, S., and Gilichinsky, D., 2007, Biogeochemistry of methane and methanogenic archaea in permafrost, FEMS Microbiol Ecol, 61(1):1-15.
Rousk, J., Baath, E., Brookes, P.C., Lauber, C.L., Lozupone, C., Caporaso, J.G., Knight, R. and Fierer, N., 2010, Soil bacterial and fungal communities across a pH gradient in an arable soil, ISME, 4:1340-1351.
Roxy, M.S., Sumithranand, V.B., and Renuka, G., 2014, Soil heat flux and day time surface energy balance, Earth Sys Sci, 123:741-750.
Sagot, B., Gaysinsky, M., Mehiri, M., Guigonis, J.M., Le Rudulier, D., and Alloing, G., 2010, Osmotically induced synthesis of the dipeptide N-acetylglutaminylglutamine amide is mediate by a new pathway conserved among bacteria, PNAS, 107:12652-12657.
Sattler, B., Post, B., Remains, D., Lutz, C., Lettner, H., and Psenner, R., 2012, Cold Alpine Regions, in Bell, E., (ed.) Life at Extremes: Environments, Organisms and Strategies of Survival, CAB International, pp. 138-154.
Schädel, C., Schuur, E.A.G., Bracho, R., Elberling, B., Knoblauch, C., Lee, H., Luo, Y., Shaver, G.R., Turetsky, M.R., 2014, Circumpolar assessment of permafrost C quality and its vulnerability over time using long-term incubation data, Glob Chang Biol, 20:641-652.
Schäfer, K., Zhang, T., Bruhwiler, L., Barrett, A.P., 2011, Amount and timing of permafrost carbon release in response to climate warming, Tellus B 63:165-180.
Schmidt, M.W., Torn, M.S., Sbvien, S., Dittmar, T., Guggenberger, G., Janssen, I.A., Kleber, M., Kögel-Knaber, I., Lehmann, J., Manning, D.A.C., Nannipieri, P., Rasse, P., Weiner, S., and Trumbore, S.E., 2011, Persistence of soil organic matter as an ecosystem property, Nature, 478:49- 56.
Schuur, E.A.G., Vogel, J.G., Crummer, K.G., Lee, H., Sickman, J.O., and Osterkamp, T.E., 2009, The effect of permafrost thaw on old carbon release and net carbon exchange from tundra, Nature, 459:556-559.
Serreze, M.C., and Barry, R.G., 2005, The Arctic Climate System, Cambridge University Press, New York, pp. 385.
Shanhun, F.L., Almond, P.C., Clough, T.J., and Smith, C.M.S., 2012, Abiotic processes dominate CO2 fluxed in Antarctic soils, Soil Biol Biochem, 53:99-111.
Shi, D., and Wang, D., 2005, Effects of various salt-alkaline mixed stresses on Anerolepidium Chinese (Trin.) Kitag, Plant and Soil, 271:15-26.
67 Shoun, H., Kim, D., Uchiyama, H., and Sugiyama, J., 1992, Denitrification by fungi, FEMS Microbiol Lett, 94:277-282.
Shur, Y., Hinkel, K.M., and Nelson, F.E., 2005, The Transient Layer: Implications for Geocryology and Climate-Change Science, Permafrost Perigl Process, 16:1-14.
Šimek, M., and Cooper, J.E., 2002, The influence of soil pH on denitrification: progress towards the understanding of this interaction over the last 50 years, European Journal Soil Sci, 53:345-354.
Simmons, B.L, Wall, D.H., Adams, B.J., Ayres, E., Barrett, J.E., and Virginia, R.A., 2009, Long-term experimental warming reduces soil nematode populations in the McMurdo Dry Valleys, Antarctica, Soil Biol Biochem, 41:2052-2060.
Sistla, S.A., Moore, J.C., Simpson, R.T., Gough, L., Shavers, G.R., and Schimel, J.P., 2013, Long- term warming restructures Arctic tundra without changing net soil carbon storage, Nature, 497:615-619.
Solaiman, Z., 2007, Measurement of Microbial biomass and activity in soil, in Varma, A., Oelmüller, R., (eds.) Advanced Techniqeus in Soil Microbiology, Springer-Verlag Berlin Heidelberg, V.11, pp. 201-2011.
Stendel, M., Christensen, J.H., and Petersen, D., 2008, Arctic climate and climate change with a focus on Greenland, Adv Ecol Res, 40:13-43.
Steven, B., Leveille, R., Pollard, W.H., and Whyte, L.G., 2006, Microbial ecology and biodiversity in permafrost, Extremophiles, 10:259-267.
Steven, B., Briggs, G., McKay, C.P., Pollard, W.H., Greer, C.W., and Whyte, L.G., 2007, Characterization of the microbial diversity in a permafrost sample from Canadian high Arctic using culture-dependent and culture-independent methods, FEMS Microbiol Ecol, 59:513-523.
Steven, B., Pollard, W.H., Greer, C.W., and Whyte, L. G., 2008, Microbial diversity and activity through a permafrost/ground ice core profile from the Canadian high Arctic, Environ Microbiol, 10:3388–3403.
Swingley, W.D., Blankenship, R.E., and Raymond, J., 2009, Evolutionary Relationships Among Purple Photosynthetic Bacteria and the Origin of Peoreobacterial Photosynthetic Systems, in Hunter, C.N., Daldal, F., Thurnauer, M.C., and Beatty, J.T., (eds.) The Purple Phototrophic Bacteria, Springer Netherlands, V.28, pp. 17-29.
Tarnocai, C., Canadell, J.G., Schuur, E.A.G., Kuhry, P., and Zimov, S., 2009, Soil organic carbon pools in the northern circumpolar permafrost region, Global Biogeochemical Cycles, 23(2):1-11.
68 Taş, N., Prestat, E., Mcfarland, J.W., Wickland, K.P., Knight, R., Berhe, A.A., Jorgeson, T., Waldrop, M.P., and Jansson, J.K., Impact of fire on active layer and permafrost microbial communities and methagenomes in an upland Alaskan boreal forest, ISME J, 8:1904-1919.
Tedrow, J.C.F., 1970, Soil Investigation in Inglefield Land, Greenland, Meddel om Gronland, V.188, C.A. Reitzels Forlag, pp. 93.
Thauer, R.K., Kaster, A.K., Seedorf, H., Buckel, W., and Hedderich, R., 2008, Methanogenic archaea: ecologically relevant differences in energy conservation, Nat Rev Microbiol, 6:579–591.
Thomas, D.N., Fogg, G.E., Convey, P., Fritsen, C.H., Gili, J.-M., Gradinger, R., Laybourn-Parry, J., Reid, K., Walton, D.W.H., 2008, The Biology of Polar Regions, Oxford University Press, pp. 416.
Tiedje, J.M, 1988, Ecology of denitrification and dissimilatory nitrate reduction to ammonium, in: Zehnder, (ed.) Environmental Microbiology of Anaerobes, John Wiley and Sons, New York, pp. 179-244.
Tiedje, J.M., Smith, G.B., Simkins, S., Holben, W.E., Finney, C., and Gilichinsky, D., 1994, Recovery of DNA, denitrifiers and patterns of antibiotic sensitivity in microorganisms from ancient permafrost soils of Eastern Siberia, in Gilichinsky, D., (ed.) Viable Microorganisms in Permafrost, Russ Acad Sci, Pushchino, pp. 83-98.
Treat, C., Natali, S., Ernakovich, J., Iversen, C., Lupascu, M., McGuire, A.D., Norby, R., Chowdhury, T.R., Richter, A., Santruckova, H., Schadel, C., Schuur, T., Sloan, V., Turetsky, M., and Waldrop,
M., 2015, A pan-Arctic synthesis of CH4 and CO2 producton from anoxic soil incubations, Glob Chang Biol, 21(7):2787-2803.
Tveit, A., Schwacke, R., Svenning, M.M., and Urich, T., 2013, Organic carbon transformation in high-Arctic peat soils: key functions and microorganisms, ISME J, 7:299-311.
Ugolini, F.C., and Anderson, D.M., 1973, Ionic migration and weathering in frozen Antarctic soils, Soil Sci, 115:461-470.
Van der Wal, R., and Hessen, D.O., 2009, Analogous aquatic and terrestrial food webs in the high Arctic: The structuring force of a harsh climate, Plant Ecol Eco Sys, 11:231-240.
Van Everdingen, R., 2005, Multi-language Glossary of Permafrost and Related Ground-ice Terms, IPA, pp. 159.
Van Horn, D.J., Okle, J.G., Buelow, H.N., Gooseff, M.N., Barrett, J.E., and Takacs-Vesbach, C.D., 2014, Soil Microbial Response to Increased Moisture and Organic Resources along A Salinity Gradient in a Polar Desert, Appl Environ Microbiol, 80:3034-3043.
69 Verstraete, W., and Focht, D.D., 1977, Biochemical Ecology of Nitrification and Denitrification, in Alexander, M., (ed.) Advances in Microbial Ecology, Plenum Press New York, V.1, pp. 135-214.
Vishnivetskaya, T., Kathariou, S., McGrath, J., Gilichinsky, D., and Tiedje, J., 2000, Low-temperature recovery strategies for the isolation of bacteria from ancient permafrost sediments, Extremophiles, 3:165-173.
Vorobyova, E., Soina, V., Gorlenko, M., Minkovskaya, N., Zalinova, N., Mamukelashvili, A., Gilichinsky, D., Rivkina, E., and Vishnivetskaya, T., 1997, The deep cold biosphere: facts and hypothesis, FEMS Microbiol Rev, 20:277–290.
Vorobyova, E., Minkovsky, N., Mamuhelashvili, A., Zvyagintsev, D., Sonia, V., Polanskaya, L., and Gilichinsky, D., 2001, Microorganisms and biomarkers in permfrost, in Paepe, R., Melnikov, V.P., Van Overloop, E., and Gorokhov, V.D., (eds.) Permafrost response on economic development, environmental security and natural resources, Kluwer Academic Publishers, Springer Netherlands, V.76, pp. 527-541.
Waldrop, M.P., Wickland, K.P., White, R.III, Berhe, A.A., Harden, J.W., and Romanovsky, V.E., 2010, Molecular investigations into a globally important carbon pool: permafrost-protected carbon in Alaskan soils, Glob Chang Biol, 16:2543-2554.
Wagner, D., 2008, Microbial Communities and Processes in Arctic Permafrost Environments, in Dion, P., and Nautiyal, C.S., (eds.) Microbiology of Extreme Soils, Springer-Verlag Berlin Heidelberg, V.13, pp. 374.
Wallenstein, M.D., McMahon, S., and Schimel, J., 2007, Bacterial and fungal community structure in Arctic tundra tussock and shrub soils, FEMS Microbiol Ecol, 59:428-435.
White, T. J., Bruns, T., Lee, S., and Taylor, J.W., 1990, Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics, in Innis, M.A., Gelfand, D.H., Sninsky, J.J., and White, T.J., (eds.), PCR Protocols: A Guide to Methods and Applications , Academic Press, Inc., New York, pp. 315-322.
Wild, B., Schnecker, J., Alves, R.J.E., Barsukov, P., Bárta, J., Čapek, P., Gentsch, N., Gittel, A., Guggenberger, G., Lashchinskiy, N., Mikutta, R., Rusalimova, O., Šantrůčková, H., Shibistova, O., Urich, T., Watzka, M., Zrazhevskaya, G., and Richter, A., 2014, Input of easily available organic C and N stimulates microbial decomposition of soil organic matter in arctic permafrost soil, Soil Biol Biochm, 75:143-151.
Wilhelm, R.C., Radtke, K.J., Mykytczuk, N.C., Greer, C.W., and Whyte, L.G., 2012, Life at the wedge: the activity and diversity of Arctic ice wedge microbial communities, Astrobiology, 12:347- 360.
70 Willerslev, E., Hansen, A.J., Rønn, R., Brand, T.B., Barnes, I., Wiuf, C., Gilichinsky, D., Mitchell, D., and Cooper, A., 2004, Long-term persistence of bacterial DNA, Curr Biol, 14:R9–R10.
Yan, N., Marschner, P., Cao, W., Zuo, C., and Qin, W., 2015, Influence of salinity and water content on soil microorganisms, International Soil Water Conserv Res, 3:316-323.
Yergeau, E., Hogues, H., Whyte, L.G., and Greer, C.W., 2010, The functional potential of high Arctic permafrost revealed by metagenomic sequencing, qPCR and microarray analyses, ISME, 4:1206- 1214.
Yu, K., Faulkner, S.P., and Baldwin, M.J., 2008, Effect of hydrological conditions on nitrous oxide, methane, and carbon dioxide dynamics in a bottomland hardwood forest and its implication for soil carbon sequestration, Glob Chang Biol, 14:798-812.
Zahran, H.H., 1997, Diversity, adaptation and activity of the bacterial flora in saline environments, Biol Fertil Soils, 25:211-223.
Zhang, Y., Chen, W., and Riseborough, D.W., 2008, Transient projections of permafrost distribution in Canada during the 21st century under scenarios of climate change, Glob Planet Chang, 60:443– 456.
Zimov, S.A., Schuur, E.A.G., and Chapin, F.S., 2006, Permafrost and the global carbon budget, Science, 312(5780):1612-1613.
Zimov, N.S., Zimov, S.A., Zimova, A.E., Zimova, G.M., Churprynin, V.I., Chapin III, F.S., 2009, Carbon storage in permafrost and soils of the mammoth tundra-steppe biome: Role in the global carbon budget, The Cryosphere, 36(2):L02502.
Zinger, L., Lejon, D.P.H., Baptist, F., Bouasria, A., Aubert, S., Geremia, R.A. and Choler, P., 2011, Contrasting diversity patterns of crenarchaeal, bacterial and fungal soil communities in an alpine landscape, PLoS ONE, 6(5):e19950.
71
10 APPENDIX
72
APPENDIX 1
The forward primer was 515F and the reverse primer was 806R. There were 12 tagged primers of each and they were mixed according to the table below. The colon “mix” indicates the mixture. “B0101” should be interpreted as forward primer 515F_01 was mixed with reverse primer 806R_01. “B0102” should be interpreted as forward primer 515F_01 was mixed with reverse primer 806R_02, and so on.
Table 1A: Primer mixtures used for tagging of PCR products for sample identification in Illumina MiSeq sequencing data.
Sample Replicate Mix Sequence of Forward Sequence of Reverse 0-10 cm Rep1 B0101 ACGTAGTGTGCCAGCMGCCGCGGTAA ACGTACCGGACTACHVGGGTWTCTAAT 0-10 cm Rep2 B0102 ACGTAGTGTGCCAGCMGCCGCGGTAA TATGCCGGACTACHVGGGTWTCTAAT 0-10 cm Rep3 B0103 ACGTAGTGTGCCAGCMGCCGCGGTAA ACACGCCGGACTACHVGGGTWTCTAAT 0-10 cm Rep4 B0104 ACGTAGTGTGCCAGCMGCCGCGGTAA GTAGCCGGACTACHVGGGTWTCTAAT 20-30 cm Rep1 B0105 ACGTAGTGTGCCAGCMGCCGCGGTAA CTGATACCGGACTACHVGGGTWTCTAAT 20-30 cm Rep2 B0106 ACGTAGTGTGCCAGCMGCCGCGGTAA CGATACCCGGACTACHVGGGTWTCTAAT 20-30 cm Rep3 B0107 ACGTAGTGTGCCAGCMGCCGCGGTAA TGCTCCGGACTACHVGGGTWTCTAAT 20-30 cm Rep4 B0108 ACGTAGTGTGCCAGCMGCCGCGGTAA CGCAGACCGGACTACHVGGGTWTCTAAT 40-50 cm Rep1 B0109 ACGTAGTGTGCCAGCMGCCGCGGTAA GACGCACCGGACTACHVGGGTWTCTAAT 40-50 cm Rep2 B0110 ACGTAGTGTGCCAGCMGCCGCGGTAA AGATCCGGACTACHVGGGTWTCTAAT 40-50 cm Rep3 B0111 ACGTAGTGTGCCAGCMGCCGCGGTAA ACTACACCGGACTACHVGGGTWTCTAAT 40-50 cm Rep4 B0112 ACGTAGTGTGCCAGCMGCCGCGGTAA GATCTCCGGACTACHVGGGTWTCTAAT 50-60 cm Rep1 B0201 TATGGTGTGCCAGCMGCCGCGGTAA ACGTACCGGACTACHVGGGTWTCTAAT 50-60 cm Rep2 B0202 TATGGTGTGCCAGCMGCCGCGGTAA TATGCCGGACTACHVGGGTWTCTAAT 50-60 cm Rep3 B0203 TATGGTGTGCCAGCMGCCGCGGTAA ACACGCCGGACTACHVGGGTWTCTAAT 50-60 cm Rep4 B0204 TATGGTGTGCCAGCMGCCGCGGTAA GTAGCCGGACTACHVGGGTWTCTAAT
73 60-70 cm Rep1 B0205 TATGGTGTGCCAGCMGCCGCGGTAA CTGATACCGGACTACHVGGGTWTCTAAT 60-70 cm Rep2 B0206 TATGGTGTGCCAGCMGCCGCGGTAA CGATACCCGGACTACHVGGGTWTCTAAT 60-70 cm Rep3 B0207 TATGGTGTGCCAGCMGCCGCGGTAA TGCTCCGGACTACHVGGGTWTCTAAT 60-70 cm Rep4 B0208 TATGGTGTGCCAGCMGCCGCGGTAA CGCAGACCGGACTACHVGGGTWTCTAAT 70-80 cm Rep1 B0209 TATGGTGTGCCAGCMGCCGCGGTAA GACGCACCGGACTACHVGGGTWTCTAAT 70-80 cm Rep2 B0210 TATGGTGTGCCAGCMGCCGCGGTAA AGATCCGGACTACHVGGGTWTCTAAT 70-80 cm Rep3 B0211 TATGGTGTGCCAGCMGCCGCGGTAA ACTACACCGGACTACHVGGGTWTCTAAT 70-80 cm Rep4 B0212 TATGGTGTGCCAGCMGCCGCGGTAA GATCTCCGGACTACHVGGGTWTCTAAT 80-90 cm Rep1 B0301 ACACGGTGTGCCAGCMGCCGCGGTAA ACGTACCGGACTACHVGGGTWTCTAAT 80-90 cm Rep2 B0302 ACACGGTGTGCCAGCMGCCGCGGTAA TATGCCGGACTACHVGGGTWTCTAAT 80-90 cm Rep3 B0303 ACACGGTGTGCCAGCMGCCGCGGTAA ACACGCCGGACTACHVGGGTWTCTAAT 80-90 cm Rep4 B0304 ACACGGTGTGCCAGCMGCCGCGGTAA GTAGCCGGACTACHVGGGTWTCTAAT 90-100 cm Rep1 B0305 ACACGGTGTGCCAGCMGCCGCGGTAA CTGATACCGGACTACHVGGGTWTCTAAT 90-100 cm Rep2 B0306 ACACGGTGTGCCAGCMGCCGCGGTAA CGATACCCGGACTACHVGGGTWTCTAAT 90-100 cm Rep3 B0307 ACACGGTGTGCCAGCMGCCGCGGTAA TGCTCCGGACTACHVGGGTWTCTAAT 90-100 cm Rep4 B0308 ACACGGTGTGCCAGCMGCCGCGGTAA CGCAGACCGGACTACHVGGGTWTCTAAT 100-110 cm Rep1 B0309 ACACGGTGTGCCAGCMGCCGCGGTAA GACGCACCGGACTACHVGGGTWTCTAAT 100-110 cm Rep2 B0310 ACACGGTGTGCCAGCMGCCGCGGTAA AGATCCGGACTACHVGGGTWTCTAAT 100-110 cm Rep3 B0311 ACACGGTGTGCCAGCMGCCGCGGTAA ACTACACCGGACTACHVGGGTWTCTAAT 100-110 cm Rep4 B0312 ACACGGTGTGCCAGCMGCCGCGGTAA GATCTCCGGACTACHVGGGTWTCTAAT 110-120 cm Rep1 B0401 GTAGGTGTGCCAGCMGCCGCGGTAA ACGTACCGGACTACHVGGGTWTCTAAT 110-120 cm Rep2 B0402 GTAGGTGTGCCAGCMGCCGCGGTAA TATGCCGGACTACHVGGGTWTCTAAT 110-120 cm Rep3 B0403 GTAGGTGTGCCAGCMGCCGCGGTAA ACACGCCGGACTACHVGGGTWTCTAAT
74 110-120 cm Rep4 B0404 GTAGGTGTGCCAGCMGCCGCGGTAA GTAGCCGGACTACHVGGGTWTCTAAT 120-130 cm Rep1 B0405 GTAGGTGTGCCAGCMGCCGCGGTAA CTGATACCGGACTACHVGGGTWTCTAAT 120-130 cm Rep2 B0406 GTAGGTGTGCCAGCMGCCGCGGTAA CGATACCCGGACTACHVGGGTWTCTAAT 120-130 cm Rep3 B0407 GTAGGTGTGCCAGCMGCCGCGGTAA TGCTCCGGACTACHVGGGTWTCTAAT 120-130 cm Rep4 B0408 GTAGGTGTGCCAGCMGCCGCGGTAA CGCAGACCGGACTACHVGGGTWTCTAAT 140-150 cm Rep1 B0409 GTAGGTGTGCCAGCMGCCGCGGTAA GACGCACCGGACTACHVGGGTWTCTAAT 140-150 cm Rep2 B0410 GTAGGTGTGCCAGCMGCCGCGGTAA AGATCCGGACTACHVGGGTWTCTAAT 140-150 cm Rep3 B0411 GTAGGTGTGCCAGCMGCCGCGGTAA ACTACACCGGACTACHVGGGTWTCTAAT 140-150 cm Rep4 B0412 GTAGGTGTGCCAGCMGCCGCGGTAA GATCTCCGGACTACHVGGGTWTCTAAT 150-170 cm Rep1 B0501 CTGATAGTGTGCCAGCMGCCGCGGTAA ACGTACCGGACTACHVGGGTWTCTAAT 150-170 cm Rep2 B0502 CTGATAGTGTGCCAGCMGCCGCGGTAA TATGCCGGACTACHVGGGTWTCTAAT 150-170 cm Rep3 B0503 CTGATAGTGTGCCAGCMGCCGCGGTAA ACACGCCGGACTACHVGGGTWTCTAAT 150-170 cm Rep4 B0504 CTGATAGTGTGCCAGCMGCCGCGGTAA GTAGCCGGACTACHVGGGTWTCTAAT 170-180 cm Rep1 B0505 CTGATAGTGTGCCAGCMGCCGCGGTAA CTGATACCGGACTACHVGGGTWTCTAAT 170-180 cm Rep2 B0506 CTGATAGTGTGCCAGCMGCCGCGGTAA CGATACCCGGACTACHVGGGTWTCTAAT 170-180 cm Rep3 B0507 CTGATAGTGTGCCAGCMGCCGCGGTAA TGCTCCGGACTACHVGGGTWTCTAAT 170-180 cm Rep4 B0508 CTGATAGTGTGCCAGCMGCCGCGGTAA CGCAGACCGGACTACHVGGGTWTCTAAT 180-190 cm Rep1 B0509 CTGATAGTGTGCCAGCMGCCGCGGTAA GACGCACCGGACTACHVGGGTWTCTAAT 180-190 cm Rep2 B0510 CTGATAGTGTGCCAGCMGCCGCGGTAA AGATCCGGACTACHVGGGTWTCTAAT 180-190 cm Rep3 B0511 CTGATAGTGTGCCAGCMGCCGCGGTAA ACTACACCGGACTACHVGGGTWTCTAAT 180-190 cm Rep4 B0512 CTGATAGTGTGCCAGCMGCCGCGGTAA GATCTCCGGACTACHVGGGTWTCTAAT 190-200 cm Rep1 B0601 CGATACGTGTGCCAGCMGCCGCGGTAA ACGTACCGGACTACHVGGGTWTCTAAT 190-200 cm Rep2 B0602 CGATACGTGTGCCAGCMGCCGCGGTAA TATGCCGGACTACHVGGGTWTCTAAT
75 190-200 cm Rep3 B0603 CGATACGTGTGCCAGCMGCCGCGGTAA ACACGCCGGACTACHVGGGTWTCTAAT 190-200 cm Rep4 B0604 CGATACGTGTGCCAGCMGCCGCGGTAA GTAGCCGGACTACHVGGGTWTCTAAT 200-210 cm Rep1 B0605 CGATACGTGTGCCAGCMGCCGCGGTAA CTGATACCGGACTACHVGGGTWTCTAAT 200-210 cm Rep2 B0606 CGATACGTGTGCCAGCMGCCGCGGTAA CGATACCCGGACTACHVGGGTWTCTAAT 200-210 cm Rep3 B0607 CGATACGTGTGCCAGCMGCCGCGGTAA TGCTCCGGACTACHVGGGTWTCTAAT 200-210 cm Rep4 B0608 CGATACGTGTGCCAGCMGCCGCGGTAA CGCAGACCGGACTACHVGGGTWTCTAAT 210-220 cm Rep1 B0609 CGATACGTGTGCCAGCMGCCGCGGTAA GACGCACCGGACTACHVGGGTWTCTAAT 210-220 cm Rep2 B0610 CGATACGTGTGCCAGCMGCCGCGGTAA AGATCCGGACTACHVGGGTWTCTAAT 210-220 cm Rep3 B0611 CGATACGTGTGCCAGCMGCCGCGGTAA ACTACACCGGACTACHVGGGTWTCTAAT 210-220 cm Rep4 B0612 CGATACGTGTGCCAGCMGCCGCGGTAA GATCTCCGGACTACHVGGGTWTCTAAT 220-230 cm Rep1 B0701 TGCTGTGTGCCAGCMGCCGCGGTAA ACGTACCGGACTACHVGGGTWTCTAAT 220-230 cm Rep2 B0702 TGCTGTGTGCCAGCMGCCGCGGTAA TATGCCGGACTACHVGGGTWTCTAAT 220-230 cm Rep3 B0703 TGCTGTGTGCCAGCMGCCGCGGTAA ACACGCCGGACTACHVGGGTWTCTAAT 220-230 cm Rep4 B0704 TGCTGTGTGCCAGCMGCCGCGGTAA GTAGCCGGACTACHVGGGTWTCTAAT 230-240 cm Rep1 B0705 TGCTGTGTGCCAGCMGCCGCGGTAA CTGATACCGGACTACHVGGGTWTCTAAT 230-240 cm Rep2 B0706 TGCTGTGTGCCAGCMGCCGCGGTAA CGATACCCGGACTACHVGGGTWTCTAAT 230-240 cm Rep3 B0707 TGCTGTGTGCCAGCMGCCGCGGTAA TGCTCCGGACTACHVGGGTWTCTAAT 230-240 cm Rep4 B0708 TGCTGTGTGCCAGCMGCCGCGGTAA CGCAGACCGGACTACHVGGGTWTCTAAT 240-250 cm Rep1 B0709 TGCTGTGTGCCAGCMGCCGCGGTAA GACGCACCGGACTACHVGGGTWTCTAAT 240-250 cm Rep2 B0710 TGCTGTGTGCCAGCMGCCGCGGTAA AGATCCGGACTACHVGGGTWTCTAAT 240-250 cm Rep3 B0711 TGCTGTGTGCCAGCMGCCGCGGTAA ACTACACCGGACTACHVGGGTWTCTAAT 240-250 cm Rep4 B0712 TGCTGTGTGCCAGCMGCCGCGGTAA GATCTCCGGACTACHVGGGTWTCTAAT 250-260 cm Rep1 B0801 CGCAGAGTGTGCCAGCMGCCGCGGTAA ACGTACCGGACTACHVGGGTWTCTAAT
76 250-260 cm Rep2 B0802 CGCAGAGTGTGCCAGCMGCCGCGGTAA TATGCCGGACTACHVGGGTWTCTAAT 250-260 cm Rep3 B0803 CGCAGAGTGTGCCAGCMGCCGCGGTAA ACACGCCGGACTACHVGGGTWTCTAAT 250-260 cm Rep4 B0804 CGCAGAGTGTGCCAGCMGCCGCGGTAA GTAGCCGGACTACHVGGGTWTCTAAT 260-270 cm Rep1 B0805 CGCAGAGTGTGCCAGCMGCCGCGGTAA CTGATACCGGACTACHVGGGTWTCTAAT 260-270 cm Rep2 B0806 CGCAGAGTGTGCCAGCMGCCGCGGTAA CGATACCCGGACTACHVGGGTWTCTAAT 260-270 cm Rep3 B0807 CGCAGAGTGTGCCAGCMGCCGCGGTAA TGCTCCGGACTACHVGGGTWTCTAAT 260-270 cm Rep4 B0808 CGCAGAGTGTGCCAGCMGCCGCGGTAA CGCAGACCGGACTACHVGGGTWTCTAAT 270-280 cm Rep1 B0809 CGCAGAGTGTGCCAGCMGCCGCGGTAA GACGCACCGGACTACHVGGGTWTCTAAT 270-280 cm Rep2 B0810 CGCAGAGTGTGCCAGCMGCCGCGGTAA AGATCCGGACTACHVGGGTWTCTAAT 270-280 cm Rep3 B0811 CGCAGAGTGTGCCAGCMGCCGCGGTAA ACTACACCGGACTACHVGGGTWTCTAAT 270-280 cm Rep4 B0812 CGCAGAGTGTGCCAGCMGCCGCGGTAA GATCTCCGGACTACHVGGGTWTCTAAT 280-290 cm Rep1 B0901 GACGCAGTGTGCCAGCMGCCGCGGTAA ACGTACCGGACTACHVGGGTWTCTAAT 280-290 cm Rep2 B0902 GACGCAGTGTGCCAGCMGCCGCGGTAA TATGCCGGACTACHVGGGTWTCTAAT 280-290 cm Rep3 B0903 GACGCAGTGTGCCAGCMGCCGCGGTAA ACACGCCGGACTACHVGGGTWTCTAAT 280-290 cm Rep4 B0904 GACGCAGTGTGCCAGCMGCCGCGGTAA GTAGCCGGACTACHVGGGTWTCTAAT 290-300 cm Rep1 B0905 GACGCAGTGTGCCAGCMGCCGCGGTAA CTGATACCGGACTACHVGGGTWTCTAAT 290-300 cm Rep2 B0906 GACGCAGTGTGCCAGCMGCCGCGGTAA CGATACCCGGACTACHVGGGTWTCTAAT 290-300 cm Rep3 B0907 GACGCAGTGTGCCAGCMGCCGCGGTAA TGCTCCGGACTACHVGGGTWTCTAAT 290-300 cm Rep4 B0908 GACGCAGTGTGCCAGCMGCCGCGGTAA CGCAGACCGGACTACHVGGGTWTCTAAT 300-310 cm Rep1 B0909 GACGCAGTGTGCCAGCMGCCGCGGTAA GACGCACCGGACTACHVGGGTWTCTAAT 300-310 cm Rep2 B0910 GACGCAGTGTGCCAGCMGCCGCGGTAA AGATCCGGACTACHVGGGTWTCTAAT 300-310 cm Rep3 B0911 GACGCAGTGTGCCAGCMGCCGCGGTAA ACTACACCGGACTACHVGGGTWTCTAAT 300-310 cm Rep4 B0912 GACGCAGTGTGCCAGCMGCCGCGGTAA GATCTCCGGACTACHVGGGTWTCTAAT
77 310-320 cm Rep1 B1001 AGATGTGTGCCAGCMGCCGCGGTAA ACGTACCGGACTACHVGGGTWTCTAAT 310-320 cm Rep2 B1002 AGATGTGTGCCAGCMGCCGCGGTAA TATGCCGGACTACHVGGGTWTCTAAT 310-320 cm Rep3 B1003 AGATGTGTGCCAGCMGCCGCGGTAA ACACGCCGGACTACHVGGGTWTCTAAT 310-320 cm Rep4 B1004 AGATGTGTGCCAGCMGCCGCGGTAA GTAGCCGGACTACHVGGGTWTCTAAT 320-330 cm Rep1 B1005 AGATGTGTGCCAGCMGCCGCGGTAA CTGATACCGGACTACHVGGGTWTCTAAT 320-330 cm Rep2 B1006 AGATGTGTGCCAGCMGCCGCGGTAA CGATACCCGGACTACHVGGGTWTCTAAT 320-330 cm Rep3 B1007 AGATGTGTGCCAGCMGCCGCGGTAA TGCTCCGGACTACHVGGGTWTCTAAT 320-330 cm Rep4 B1008 AGATGTGTGCCAGCMGCCGCGGTAA CGCAGACCGGACTACHVGGGTWTCTAAT 330-340 cm Rep1 B1009 AGATGTGTGCCAGCMGCCGCGGTAA GACGCACCGGACTACHVGGGTWTCTAAT 330-340 cm Rep2 B1010 AGATGTGTGCCAGCMGCCGCGGTAA AGATCCGGACTACHVGGGTWTCTAAT 330-340 cm Rep3 B1011 AGATGTGTGCCAGCMGCCGCGGTAA ACTACACCGGACTACHVGGGTWTCTAAT 330-340 cm Rep4 B1012 AGATGTGTGCCAGCMGCCGCGGTAA GATCTCCGGACTACHVGGGTWTCTAAT 340-350 cm Rep1 B1101 ACTACAGTGTGCCAGCMGCCGCGGTAA ACGTACCGGACTACHVGGGTWTCTAAT 340-350 cm Rep2 B1102 ACTACAGTGTGCCAGCMGCCGCGGTAA TATGCCGGACTACHVGGGTWTCTAAT 340-350 cm Rep3 B1103 ACTACAGTGTGCCAGCMGCCGCGGTAA ACACGCCGGACTACHVGGGTWTCTAAT 340-350 cm Rep4 B1104 ACTACAGTGTGCCAGCMGCCGCGGTAA GTAGCCGGACTACHVGGGTWTCTAAT Negative Rep1 B1105 ACTACAGTGTGCCAGCMGCCGCGGTAA CTGATACCGGACTACHVGGGTWTCTAAT Negative Rep2 B1106 ACTACAGTGTGCCAGCMGCCGCGGTAA CGATACCCGGACTACHVGGGTWTCTAAT Negative Rep3 B1107 ACTACAGTGTGCCAGCMGCCGCGGTAA TGCTCCGGACTACHVGGGTWTCTAAT Negative Rep4 B1108 ACTACAGTGTGCCAGCMGCCGCGGTAA CGCAGACCGGACTACHVGGGTWTCTAAT Negative Rep5 B1109 ACTACAGTGTGCCAGCMGCCGCGGTAA GACGCACCGGACTACHVGGGTWTCTAAT Negative Rep6 B1110 ACTACAGTGTGCCAGCMGCCGCGGTAA AGATCCGGACTACHVGGGTWTCTAAT Negative Rep7 B1111 ACTACAGTGTGCCAGCMGCCGCGGTAA ACTACACCGGACTACHVGGGTWTCTAAT
78 Appendix 2
The figure below shows the correlation between CO2 production rate and number of 16S rRNA gene copies obtained from the different depths on a logarithm scale. No tendency can be seen between the number of genes present and the amount of gas produced between the three different soil layers.
Figure 1A: Visualization of the correlation between 16S rRNA gene copies and CO2 production at the different depths. Blue ♦ represents the AL, purple ♦ represents the YP and green ♦ represents the OP.
79 Appendix 3
The figure below shows the number of sequences obtained at each depth. As it can be seen there are great variations throughout the soil core. The number was highest at 320-330 cm depth, but has also the largest error bars, due to great variations between each replicate.
Figure 2A: Total number of sequences at each depth including standard error of the mean.
80