Importance of winter climate and soil frost for dissolved organic carbon (DOC) in boreal forest soils and streams: - implications for a changing climate

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Importance of winter climate and soil frost for dissolved organic carbon (DOC) in boreal forest soils and streams

- implications for a changing climate

Mahsa Haei

Department of Ecology and Environmental Science Umeå University

Umeå 2011

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Printed by: VMC-KBC, Umeå University Umeå, Sweden 2011

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List of papers

This thesis is based on the following papers, which are referred to in the text by the corresponding Roman numerals:

Paper I: Haei, M., M. G. Öquist, I. Buffam, A. Ågren, P. Blomkvist, K.

Bishop, M. Ottosson Löfvenius and H. Laudon (2010), Cold winter soils enhance dissolved organic carbon concentrations in soil and stream water.

Geophysical Research Letters, 37, L08501, doi: 10.1029/2010GL042821.

Paper II: Ågren, A., M. Haei, S. J. Köhler, K. Bishop and H. Laudon (2010), Regulation of stream water dissolved organic carbon (DOC) concentrations during snowmelt; the role of discharge, winter climate and memory effects. Biogeosciences, 7, 1-13, doi: 10.5194/bg-7-2901-2010.

Paper III: Haei M., M. G.Öquist, U. Ilstedt and H. Laudon (2010), The influence of soil frost on the quality of dissolved organic carbon in a boreal forest soil - combining field and laboratory experiments. Biogeochemistry, doi: 10.1007/s10533-010-9534-2.

Paper IV: Haei M., J. Rousk, U. Ilstedt, M. G. Öquist, E. Bååth and H.

Laudon (2011), Effects of soil frost on growth, composition and respiration of the soil microbial decomposer community. Under review in Soil Biology and Biochemistry.

Reproduced with the kind permission of American Geophysical Union (Paper I) and Springer (Paper III).

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Introduction

Dissolved organic carbon (DOC), the carbon fraction of dissolved organic matter, is operationally defined as all organic molecules which pass through filters with a mesh size ≤ 0.45 µm. DOC plays a fundamental role in biogeochemical processes as it affects pH (Hruska et al., 2003), acts as a source of energy and nutrients (Leenheer et al., 2003; Steinberg et al., 2006;

Berggren et al., 2010), and serves as a strong complexing agent for metals and micropollutants, thus influencing their transport (e.g. Bishop et al., 1995; Maxin et al., 1995; Dillon et al., 1997; Cory et al., 2006; Bergknut et al., 2010). In addition, DOC influences the depth of the photic zone in surface waters (Weishaar et al., 2003; Karlsson et al., 2009), affects food web structure (Jansson et al., 2007), and is important to the overall landscape carbon balance (Cole et al., 2007).

Aquatic systems receive large amounts of terrestrially derived organic carbon (Cole et al., 2007; Battin et al., 2009) which originates from litter and humus, root exudates and microbial biomass. Organic matter in soil undergoes various processes such as adsorption-desorption, precipitation- dissolution, diffusion, complexation-decomplexation and protonation- deprotonation (Kalbitz et al., 2000). Moreover, soil DOC includes a diversity of constituents that range from low-molecular weight compounds such as amino acids and carbohydrates to high molecular weight humic substances, with the latter as the predominant component (van Hees et al., 2005).

Vegetation type (Neff et al., 2002) and soil decomposer community (Lundquist et al., 1999) are important factors for production and processing of DOC in soils. In addition, soil properties such as pH and mineral composition (Kalbitz et al., 2000), and environmental conditions such as temperature and moisture content (Christ et al., 1996) can also be important factors controlling the production and fate of DOC in terrestrial ecosystems.

In the two last decades, increasing concentrations of DOC observed in streams and lakes across large parts of the northern hemisphere (Monteith et al., 2007) have motivated extensive research on the drivers of DOC loss from catchments. Statistical analyses suggest that climate variability (Prokushkin et al., 2005; Erlandsson et al., 2008),decline in acid deposition (Evans et al., 2006; Haaland et al., 2010) and land-use change(Findlay et al., 2001; Kalbitz, 2001) are all potentially responsible for this observed increase. However, the evidence to date is far from conclusive because most studies have either been based on long-term time-series of surface water chemistry or on short-term experimental work.

Mid- to high-latitude regions are characterized by a distinct winter season and periods of snow-cover. Snow-cover regulates soil biogeochemical processes during the winter by insulating the soil surface which reduces the heat loss and can control the formation of soil frost (Groffman et al., 2001; Stieglitz et al., 2003). In many seasonally snow-

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covered regions, snow provides a substantial fraction of the annual water budget, and spring snow-melt is the dominant annual hydrological event (Barnett et al., 2005). These same regions also store more than half of the global carbon (Hobbie et al., 2000; Tarnocai et al., 2009), play a major role in carbon cycling, and are particularly vulnerable to anticipated climate change (IPCC, 2007). For example, climate models for the end of the 21^st century predict the most dramatic increase in temperature and precipitation in northern latitude regions and this change is expected to occur mostly in the winter season (Christensen et al., 2007). Such changes may therefore alter the timing of snow-pack formation and melting and therefore change the duration of snow-covered periods (Mellander et al., 2007; Jylha et al., 2008). Moreover, changes in lower latitude regions may include a shift from a persistent to more intermittent snow-pack (Hayhoe et al., 2007). As a result, the soil frost regime and frequency of freeze-thaw cycles also is likely to be affected by a changing winter climate (Stieglitz et al., 2003; Henry, 2008).

The riparian zone, the last few meters of soil which water passes through before entering the adjacent stream, acts as a significant source of carbon to aquatic systems in many regions, particularly during high-flow episodes (Dosskey et al., 1994; Hinton et al., 1998; Seibert et al., 2009).

Riparian zones, which have distinct hydrological connectivity between uplands and streams, also exert a major control on runoff generation (McGuire et al., 2010) and fluxes of solutes and nutrients (Petrone et al., 2007; Lyon et al., 2011). In recently glaciated regions, riparian zones also accumulate a considerable amount of organic carbon stored as peat, which results from water-logged conditions and low decomposition rates along the stream channel (Vidon et al., 2010). Prior to entering the streams, water flowing laterally through this riparian peat tends to become concentrated with DOC (Bishop et al., 2004). Therefore, the riparian zone significantly controls stream DOC concentrations, and even small changes in riparian soil DOC production can potentially have important implications for adjacent streams.

In this thesis, I investigated the link between stream DOC concentration during the spring snow-melt period and key hydro-climatic factors.

Specifically, this work addressed the regulatory effects of winter climatic conditions on soil biogeochemistry and the direct control of riparian soils on the chemistry of adjacent streams. In addition, field and laboratory experiments were combined to study the influence of soil frost and winter climate variables on the quality and quantity of DOC in riparian soils. In the laboratory, I took a further step to explore how the activity and composition of soil microbial community respond to soil freezing and if it could be connected to frost induced changes in DOC.

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Thesis objectives and overview

In this thesis I address the following over-arching questions:

 How do winter climatic factors affect stream DOC concentrations during spring snow-melt in boreal forested streams? (Papers I and II)

 How do winter climate and soil frost affect the quality and quantity of DOC in riparian forest soils? (Papers I and III)

 How does soil freezing influence the growth and composition of the soil microbial community, and to what degree is microbial activity linked to frost-induced increases in DOC? (Paper IV)

Figure 1 summarizes the research in different papers that was carried out to answer the above questions. Arrows in the figure also serve to illustrate how these different efforts were conceptually linked in order to broadly assess the controls on spring-time DOC losses in boreal streams.

Figure 1. Brief summary of the papers included in this thesis

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Materials and methods

Study area

The studied streams and the soil manipulation area, as well as soil sampling area for the laboratory analyses, were parts of the Krycklan catchment at Svartberget Long-term Ecological Research Forest (Svartberget LTER;

64°14’N, 19°46’E), 60km northwest of Umeå, Sweden. The stream studied in Paper I drains the 50ha Svartberget catchment which is forest dominated (15% mire) (C7, Figure 2). The stream studied in Paper II, Västrabäcken (C2, Figure 2), is a small first-order stream in an entirely forested subcatchment of the Svartberget catchment (12ha). The soil frost manipulation experiment (Papers I and III) was established in the riparian zone of Västrabäcken (C2). Soil samples for laboratory experiments (Papers III and IV), were collected in the upper 30cm approximately 10m downstream the soil frost manipulation experiment along Västrabäcken.

Stream DOC, discharge and winter climate variables (1993-2007)

Streams were sampled in acid washed and stream rinsed high-density polyethylene bottles. During the spring snow-melt period, sampling was carried out weekly before 2002 and more frequently thereafter (Papers I and II). Discharge was measured using calibrated V-notch weirs located in a heated house and recorded using a Campbell Scientific data logger equipped with a pressure transducer, and hourly estimates were aggregated to generate daily flow values (Papers I and II). Specific discharge values used in Papers I and II are based on the measurements at C7 (Figure 2) and rescaled to the catchment areas in the respective streams. Winter climate data were

Figure 2. Map of the study area:

Forest dominated Svartberget catchment and its entirely forested Västrabäcken subcatchment. White area is covered with forest and the gray area is mire. Light gray lines indicate the topography (5m contour interval) and dark gray lines show the streams. Filled circles marked with C7 and C2 indicate the locations for stream water sampling for Papers I and II, respectively. Discharge was measured at C7. The asterisk indicates the location of riparian soil sampling for the laboratory studies.

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measured in an open field at the Svartberget climate reference station located 1.2km from study catchment, following the Swedish Meteorological and Hydrological Institute’s (SMHI) standards (Papers I and II).

Soil frost manipulation experiment (2003-2008)

The soil frost manipulation experiment (Paper I and III) in the riparian zone of Västrabäcken (C2, Figure 2), included triplicates of the following treatments: deep soil frost, shallow soil frost and control (Figure 3). Each treatment plot had an area of 9m², with a centre where soil measurements were conducted, located at a distance of 2.9±0.5m from the study stream.

The deep soil frost treatment plots were covered with a roof on which the snow accumulated, thereby preventing snow-pack formation and inducing deep soil frost. The shallow soil frost treatment plots were insulated with geotextile bags containing Styrofoam pellets. The control plots were exposed to ambient conditions. To ensure the hydrological balance between the treatment plots upon snow-melt, the accumulated snow on the top of the roofs was added to the ground at the end of each winter.

Figure 3. The design of the field-scale soil frost manipulation experiment with triplicates of each treatment. Photos: Peder Blomkvist.

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Suction lysimeters were installed in the centre of each plot at the depths of 10, 25, 40, 60 and 80cm. Soil water samples were collected using pre- evacuated bottles (-100kPa), 8-15 times a year with more frequent sampling during spring and summer. Soil temperature and water content (using time- domain reflectometry, TDR) were continuously recorded. Maximum soil frost depth in the treatment plots was estimated by linear interpolation between frozen and unfrozen soil layers. The soil layer at which temperature

≤ 0°C and no liquid water was detected was considered frozen, while temperature > 0°C and TDR response indicating liquid water represented unfrozen conditions (Papers I and III). Quality of DOC was assessed by calculating the carbon specific ultraviolet absorbance (SUVA₂₅₄ index calculated by dividing the absorbance at 254nm by DOC concentration) which is a commonly used indicator of the aromatic content of the organic matter (Weishaar et al., 2003; Vogt et al., 2004).

Multi-factor laboratory experiment

A series of controlled laboratory experiments were conducted to mimic natural conditions and study the effects of soil freezing and winter climate on the quality and quantity of DOC (Paper III) as well as soil microbial activity and composition (Paper IV). These experiments were carried out using a bulk riparian soil sample, collected to 30cm depth. After pretreatments in the laboratory, this bulk soil sample was split into 27 experimental sub-samples based on a central composite face-centered (CCF) design (more details on CCF design in Box 1). The CCF design was centered on three levels of four frost-related factors, namely temperature (0°C, -6°C, -12°C), water content (30%, 60% and 90% water holding capacity), freeze- thaw cycles (1, 4 and 7 cycles) and experiment duration (2, 4 and 6 months).

Box 1

CCF design used in this study is classified as a response surface method/modeling (RSM). Instead of studying the effect of several different factors individually, RSM enables the study of the integrated effects of several factors, in a particular range, on desired response(s). For some applications, RSMs can be useful to identify the optimal combination of a group of influential factors to produce a desirable level of the studied response (Box et al., 1978). Central composite face-centred design (CCF) is based on three levels of normally 2-5 factors. In a CCF design, the experimental area for three factors is a cube in which the axial points are located on the centre of each face, and together with vertices (corner points) cover the range between the lowest and highest levels of each studied factor in each dimension. For each additional factor, an extra dimension is added to the experimental design’s domain.

The design also includes replicated points, normally three centre points, at the centre of the cubic experimental area. The replicated centre points are based on the centre points of all of the applied factors (Eriksson et al., 2008).

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The 27 experimental samples were comprised of different combinations of the factors as illustrated in Figure 4. At the end of each experimental period (2-6 months), DOC was extracted and UV-absorbance was measured. In addition, fungal and bacterial growth, soil basal respiration and PLFA composition was assessed.

Figure 4. Illustration of the experimental domain and the 27 experimental samples based on central composite face-centered (CCF) design in the multi-factor laboratory experiment

The DOC extraction was performed by shaking 1-to-5 soil mass-to- water volume slurry followed by centrifugation (slight modification of Jones et al. (2006)) and filtration through 0.45µm mixed cellulose ester (MCE) filters prior to DOC analysis (Papers III and IV). In Paper IV, fungal growth was assessed by acetate (Ac) incorporation into fungal ergosterol (Newell et al., 1991; Bååth, 2001; Rousk et al., 2007; Rousk et al., 2009). Bacterial growth was estimated by incorporation of leucine (Leu) into bacteria (Kirchman et al., 1985; Bååth, 1994; Bååth et al., 2001). Soil basal respiration was measured hourly using a respirometer (Respicond IV, Nordgren Innovations AB, Djäkneboda, Sweden) (Nordgren, 1988;

Nordgren, 1992). The phospholipid fatty acid (PLFA) composition was determined according to Frostegård et al. (1993) with modifications (Nilsson et al., 2007). Sum of the concentrations of the PLFAs i15:0, a15:0, 15:0, i16:0, 16:1ω9, 16:1ω7c, 10Me16:0, cy17:0, a17:0, 18:1ω7, and cy19:0 were used to indicate the bacterial biomass, while 18:2ω6,9 was used as an indicator of fungal biomass (Frostegård et al., 1996).

Data analyses and statistics

In Paper I, partial least square (PLS) regression analysis (see more details on PLS in Box 2) was used to study the effects of variability in “winter climate”, “antecedent conditions” and “snow-melt” variables (a total of 29 descriptor variables) on “peak spring snow-melt DOC (DOCmax)”. The same

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approach was used in Paper II to study the effects of similar descriptor variables (a total of 32 variables) on i) maximum DOC concentration during spring snow-melt (DOC_M)¹, ii) average DOC concentration during spring snow-melt (DOCAVERAGE), iii) DOC concentration during the day with maximum discharge (DOC_F) and iv) overall DOC export during spring snow-melt (DOCEXP). In both cases, the goal was to evaluate the drivers of the inter-annual variation in snow-melt stream DOC concentration observed over a 15-year period. In Paper II, this analysis was complemented by removing hydrological effects using a method described by Seibert et al.

(2009): briefly, Residual DOCM, Residual DOCAVERAGE and Residual DOCF

were calculated as the difference between the observed and modeled DOC concentrations and used as indicators of variability in soil water DOC concentration. The DOC residuals were thereafter tested in response to winter climate and preceding condition variables. The analyses were evaluated at 5% (and in some cases at 10%) significance level. The final PLS models were achieved by successive refinement of the initial models and included only those variables that were both significant at the desired level and had high PLS weights. In order to interpret some of the more complex PLS models in Paper II, analyses were complemented by multiple linear regression analysis (MLR), investigating the correlation between each response variable and one representative variable from each category of descriptor variables.

In the field-scale soil frost manipulation experiment, non-parametric Mann-Whitney U-test (5% significance level) was performed to identify the significance of the soil frost treatment effects on both spring and summer DOC concentrations (Paper I) and SUVA254 (Paper III) in the soil solution samples at the five depths in the soil profiles of the treatment plots. The responses of spring and summer DOC concentration and SUVA254, respectively in Papers I and III, to variations in soil frost duration in the antecedent winter, was evaluated using linear regression analyses (5%

significance level).

In the multi-factor laboratory experiment, all of the initial models were refined by removing the non-significant factors and their two-way interactions (5% significance level). Responses of DOC concentration and SUVA₂₅₄ to the soil freezing factors and interactions were studied by creating multiple linear regression (MLR) models (PLS approach produced identical results for DOC), while the responses of fungal growth rate, bacterial growth rate, soil respiration rate and PLFA concentrations were based on PLS models. PLFA compositions were further analyzed by Principal Component Analysis (PCA) to identify the relationship between

1 Corresponds to DOCmax in Paper I

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the PLFA markers and their correlation to the experiments’ descriptor variables. Linear regression analysis was used to assess how much of the variations in fungal growth rate, bacterial growth rate and soil respiration rate could be explained by the variation in frost induced DOC concentrations. White’s test was used to detect heteroscedasticity of the residual in the linear regression analyses. Prior to all of the linear regression analyses, the normal distribution of the variables was tested using Kolmogorov-Smirnov test and data were transformed when needed.

Box 2^*

PLS stands for projection to latent structures by means of partial least squares. The PLS method describes the association between X and Y matrices by linear multivariate models. PLS method can cope with collinearity, missing values and noise in both matrices. In addition, the data are normally scaled to unit variance and mean- centered, giving equal importance to all of the variables. While constructing a PLS model, each observation is represented by one point in the X space and one point in the Y space. Since the data are normally mean-centered, all groups of the data in the X and Y spaces pass through the mean point which has been set to zero.

The first PLS component is a line in the X space and a line in the Y space fitted to best summarize the variation in X and Y, while the co-variation between the X and Y scores is maximized. Scores are obtained by projecting each observation to the component line. To better approximate the data in each space and enhance the correlation between the two matrices, more components may be added to the PLS model. The second component is orthogonal to the first one in the X matrix (it is not necessary the case in the Y space). More than two components may be added to the model. This changes the two-dimensional plane of the X and Y matrices to hyper- planes of n dimensions (n=number of components). All of the PLS components pass through the same origin as the original data vectors (zero). Additional components normally improve the ability of the model to explain the variation in the dataset which is indicated by approaching the goodness of fit (R²) to unity. However, the predictive ability of the model (indicated by goodness of prediction (Q²)) reaches a threshold while the model gets more complicated. Therefore, the number of components included in a final PLS model should ensure the optimal balance between R² and Q².

In this thesis, the significance of X variables was assessed by using PLS regression coefficients plots (based on scaled and centered variables). The size of each PLS regression coefficient indicates the changes in the corresponding Y variable when the scaled and centered X variable changes in the range of 0-1. The direct or inverse correlation between the X and Y variable is indicated by the sign of the coefficient. The statistical significance of a coefficient is indicated by confidence interval which is normally assessed at 95%. In addition, the PLS weight plots (including both X- and Y weights) were used to assess the correlation structure between X and Y variables. For each PLS component, PLS weights represent the link between the original variables and the PLS scores. In a PLS weight plot, X variables with further distance from the origin along each component are the most influential variables. In the PLS weight plot, positive or negative correlation of variables is assessed by their position relative to the origin. In order to improve the goodness of fit and goodness of prediction for a PLS model, the model can be refined by excluding the insignificant variables and/or variables with low PLS weights.

* (Eriksson et al., 2008)

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Results and discussions

Role of winter climate in regulating stream DOC

Large inter-annual variation was observed in stream DOC concentration over the fifteen year period of record (Papers I and II). For example, measures of DOC in Paper II ranged between 11.7-26.1 mg l^-1 for DOCAVERAGE, 17.7-30.1 mg l^-1 for DOCM and 15.6-19.3 mg l^-1 for DOCF. Three major groups of factors showed to be influential for regulating these patterns of stream-water DOC during spring snow-melt: (i) prior DOC export in the preceding summer/fall, ii) the intensity and duration of the snow-melt hydrograph, and iii) the severity of the antecedent winter climate conditions. Specifically, in the studies of stream chemistry, it was shown that higher prior export of DOC and a less intensive snow-melt hydrograph were both followed by lower concentrations during spring snow-melt period.

Among the identified descriptor variables, the followings were significant in both studies (Papers I and II): total discharge and DOC export during preceding summer/fall, and the duration of snow-melt rising limb representing the spring flood event. In general, these results are consistent with previous studies that have shown hydrology to be an important controlling factor for stream DOC concentrations (Hornberger et al., 1994;

Dawson et al., 2008; Köhler et al., 2009).

In addition to these hydrologic effects, longer winters resulted in higher peak DOC concentration during the following spring snow-melt. This was represented in Papers I and II by the two factors: “ending date of antecedent winter” and “number of preceding winter’s days with subzero air temperature”. Furthermore, in the first study focusing on the larger, forest- dominated catchment (C7, Paper I), two variables representing accumulated winter temperatures in air and at 10cm soil depth exhibited significant influences on spring snow-melt DOC_max. The additional DOC measures in the smaller, entirely forested site (C2, Paper II) exhibited significant responses to the same categories of factors as did DOC_M. In Paper II, focusing on the DOC residuals that correct for hydrological effects, it was shown that the variation in snow-melt DOC in some years was indeed explained by discharge, while in other years other factors controlled the variation (as indicated by a large residual DOC), which could largely be explained by longer and colder winters and secondarily by higher discharge and export during the antecedent seasons.

Impacts of soil frost on riparian soil water DOC

The soil frost manipulation experiment in the riparian zone of Västrabäcken induced distinct treatment effects on the minimum winter soil temperature (±SD) (-5.2°C (1.1), -2.2°C (1.0), -0.2°C (0.4)), maximum soil frost depth (±SD) (49 (6), 29 (3), 4 (5)cm), and soil water DOC concentrations (Figure 5) during spring and summer, at 10cm soil depth (Paper I). The mean spring

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and summer DOC concentration at 10cm depth was significantly different among treatments, with deep soil frost having the highest value and the shallow soil frost the lowest. The DOC concentrations under the shallow soil frost treatments were significantly lower than either the deep soil frost treatment or controls at 25cm soil depth.

When splitting the study period to snow-melt/spring and summer/fall, the deep soil frost treatment still showed a significant increase as compared to either shallow soil frost or control treatments at 10cm soil depth. DOC in the deep soil

frost treatment exhibited higher lability, as indicated by lower SUVA₂₅₄ values, in the upper soil horizons (Paper III). Mean SUVA₂₅₄ at deep soil frost (3.6 l mg^-1 C m^-1) was significantly lower than the control treatment at 10cm depth (4.5 l mg^-1 C m^-1). At 25cm depth, mean SUVA₂₅₄ at deep soil frost was significantly lower than control and shallow soil frost.

In the field, soil frost manipulation resulted in different long winter periods in the soil (10cm) as defined by the number of days with subzero temperatures. Length of winter appeared to largely explain the variations in both quantity (Figure 6) and quality of soil water DOC during the following spring and summer. The DOC concentration was positively (Paper I) and SUVA254 (Paper III) was negatively correlated with the length of winter. In other words, longer winters led to higher concentrations and higher lability of DOC. This result, regarding soil water DOC concentration, was also supported by the significant correlation between residual DOCM and duration of winter (assessed by number of days with sub-zero air temperature) (Paper II). Residual DOCM whichindicated the variability in soil water DOC concentration (inferred from stream DOC) after removing the effect of hydrology, revealed a similar positive and significant response to the duration of winter (R²=0.52 and p=0.003; Figure 6).

Figure 5. Soil frost treatment effect on riparian soil water DOC concentrations during spring and summer at the depth of 10cm

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In the laboratory experiment, low incubation temperature was the main influential factor for changing the amount and composition of DOC, with lower temperatures resulting in more DOC and higher DOC lability (Paper III). Yet, in the field study, no significant correlation was found between the soil temperature and either DOC or SUVA254. However, the soil samples in the laboratory experiment were constantly exposed to low temperatures for 2-6 months and the lower levels of temperature applied in the laboratory experiment produced harsher conditions compared to what soils in the field experienced. On the other hand, frost duration did not turn out to be significant for DOC or SUVA254 in the laboratory (Table I) (Paper III), but significantly increased the DOC concentration and lability at 10cm depth in the field study. However, the ‘frost durations’ applied in these two different studies do not necessary imply the same conditions. Frost duration as used in the field study (Papers I and III) included any winter soil temperature below 0°C, which on average declined to -5.2°C under the deep soil frost treatment plots at 10cm depth, while incubation duration in the laboratory included periods of 2, 4 and 6 months at constant temperatures of 0°C, -6°C and -12°C (Paper III). In addition, in the laboratory, water content exhibited significant effects on DOC and SUVA₂₅₄(Table 1); DOC concentration and lability were higher in the moister soils (Paper III). We also observed a shift towards lower C:N ratio at lower incubation temperatures which could indicate a microbial origin of the organic matter (Paper III). Frequency of freeze-thaw cycles was not a significant influential factor.

The existing literature on the effects of soil frost and freeze-thaw events on soil solution DOC provides inconclusive results. However, because of various methods used in different studies, it is hard to draw general conclusions (Henry, 2007). In the northern hardwood forests of New Hampshire, USA, no soil freezing effect was detected on the soil solution DOC after a two-year snow removal treatment (Fitzhugh et al., 2001).

However, Groffman et al. (2010) found higher DOC concentration as Figure 6. Soil solution DOC

concentration during spring and summer in response to the

‘duration of antecedent winter’

in soil, as well as the response of residual DOCM to the length of winter as assessed by the number of days with sub-zero air temperatures. Duration of winter in soil was defined as the number of days with sub-zero temperature at 10cm depth.

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affected by soil frost in a two-year study performed at a later occasion in the same area, but the changes were not consistently significant. In Norwegian heathland soils, soil frost was shown to increase the soil solution DOC and shift the SUVA₂₅₄ towards lower values in both field (Austnes et al., 2008b), and laboratory experiments with two month permanent soil frost at -5°C (Austnes et al., 2008a; Vestgarden et al., 2009). In a study of forest soils in Germany, no response to soil frost was detected in the field (Hentschel et al., 2009), while a two-week permanent soil frost in the laboratory increased the DOC concentrations at -8°C and -13°C, but not at -3°C, and did not lead to higher DOC lability (Hentschel et al., 2008). Previous investigations have reported higher DOC concentrations after freeze-thawing at -5°C (Grogan et al., 2004; Austnes et al., 2008a), while no effects of freeze-thaw cycles were found on the quality of DOC (Austnes et al., 2008a; Hentschel et al., 2008).

In contrast to our study which has been going on for eight years (Paper I), most previous field investigations included short-term manipulation studies (one to two years) with higher potential of experimental artifacts which did not allow the study of more long-term effects. In addition, we maintained the water balance among the treatments in our field study.

Lysis of microbial cells (Soulides et al., 1961), damage to fine roots (Tierney et al., 2001), physical disruption of the soil aggregates (Oztas et al., 2003) and desorption of previously adsorbed organic material (Yurova et al., 2008) are among the potential driving mechanisms for increase in DOC concentrations during the soil frost periods. Fine root mortality (Tierney et al., 2001) and plant root injuries (Gaul et al., 2008) caused by frost and low temperatures may also result in a boost of more labile organic matter in the form of simple sugars and amino acids during winter (Scott-Denton et al., 2006). In addition, changes in the winter soil microbial activity and composition (Clein et al., 1995; Bölter et al., 2005) as well as alteration of vegetation cover and performance by frost and winter climate (Kreyling, 2010) may also have implications for the release of DOC (Vestgarden et al., 2009). However, the exact contributions of the potential mechanisms are as yet unclear. The significant interactions of factors in our laboratory experiment (Table 1) demonstrate the complexity of the conditions affecting the release and composition of DOC and highlight the need for simultaneous study of the influential factors and their integrated effects.

Impacts of freezing on soil microbial community

In contrast to most of the previous studies applying biomass-based assessments (e.g. Feng et al., 2007; Schmitt et al., 2008), this study focused on the results of soil freezing on the actively growing fungi and bacteria.

Fungal and bacterial growth rates responded oppositely to constant low incubation temperatures, ranging from -12°C to 0°C, in our laboratory experiment (Table 1). In the samples kept at the highest temperature (0°C),

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Table 1. PLS regression coefficients for the scaled and centered variables studied in the multi-factor laboratory study in response to the four freezing related factors and their two-way interactions. The values for the significant factors/interactions are presented.

(IT: incubation temperature, WC: water content, D: experiment duration, FTC: freeze- thaw cycles)

[DOC] SUVA254 Fungal growth rate

Bacterial growth

rate

Soil respiration

rate

Total [PLFA]¹ Factors

IT -0.11 +0.16 -0.24 +0.24 - +0.04

WC +0.07 +0.04 - - +0.08 -

D - - - -0.22 -0.06 -0.07

FTC - - - - - -

Two-way interactions

IT*WC +0.04 +0.03 - - - -

IT*D - +0.02 - - - -

IT*FTC - -0.02 - - - -

WC*D - +0.06 +0.12 +0.03 -

WC*FTC +0.04 -0.03 - - - -

D*FTC - - - - - -

1The responses of total, fungal and bacterial [PLFA] were similar and therefore the regression coefficients for total [PLFA] is only shown.

fungal growth rate increased, while the bacterial growth rate declined compared to the lowest applied temperature (-12°C). In addition, longer experiment durations led to lower bacterial growth rates, while it did not appear to be important for the fungal growth rate. Interaction between the duration of experiment and soil water content was significantly influential for both fungal and bacterial growth rates (Table 1). Longer experiment durations decreased the soil respiration rate in the drier soils to a much higher extent than in the moist soils (almost no effect was observed in the wettest soils). The overall responses of the fungal and bacterial growth rates to frost-induced DOC were also reverse: fungal growth rate responded positively to increased DOC while the bacterial growth rate declined. Soil respiration rate was also enhanced by frost induced DOC.

Considering the similar type of limitations caused by freezing-thawing and drying-rewetting, our results are in line with Bapiri et al. (2010) and changes in the fungal:bacterial ratio could be explained by similar mechanisms such as restrictions on water availability and substrate diffusion.

This suggests that fungi may be better competitors than bacteria under fluctuating water or nutrient conditions. In addition, bacterial access to substrate is restricted to the immediate environment adjacent to their cell walls (McMahon et al., 2009). The tendency for the respiration to be positively correlated with DOC has been previously reported (Michaelson et

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al., 2003). However, it is still not well established if the DOC is the driving factor for increasing respiration or vice versa.

Prolongation of the incubation duration from two to six months and alteration of incubation temperature towards the lowest temperature (-12°C) led to ~30% decline in total PLFA concentrations. This is in line with the research by Schmitt et al. (2008) and could be caused by frost damage to a part of the microbial community. We also observed a clear shift in the PLFA composition at 0°C compared with the lower temperatures, with unsaturated PLFAs associated with fungi and gram negative bacteria being more abundant at 0°C. Since rates of changes in the membrane composition can to a large extent be reduced by freezing (Drotz et al., 2010), observed PLFA patterns at 0°C could partly be due to the most optimal conditions at constant non-freezing temperature in which microorganism could adjust their cell membranes due to phenotypic plasticity (Russell and Fukunaga, 1990;

Pettersson et al., 2003).

Concluding remarks

The results presented in this thesis demonstrate a strong link between winter climatic conditions and concentrations of stream DOC. I also show that long-term soil frost enhances the soil solution DOC concentration, which based on the hydrological conditions, might have implications for the water quality in adjacent streams. In addition, these results highlight that changes in DOC in a changing future climate will be affected by a combination of mechanisms in the soil and processes occurring at the soil-stream interface.

Predicted increase in precipitation may lead to wetter soils in which the rate of freeze-thawing in the beginning and end of winter may be changed. How the soil temperature might change depends on the integrated effect of changes in air temperature and the thickness of snow-pack which acts as the soil insulator. Based on my findings, a changing climate with colder soils and deeper soil frost produces more DOC. However, if larger amounts of DOC are exported to streams in the fall and winter, as the result of wetter soil conditions caused by higher precipitation, then stream DOC concentrations during the spring snow-melt might not be as high. On the other hand, the reduction in the amount of water provided by a thinner snow- pack might export a smaller amount of the soil DOC. Thus even though colder soils might produce higher DOC, shorter and warmer winters may be followed by reduced stream DOC concentrations. Based on the findings of this thesis, changes in the frequency of freeze-thaw events are likely not as important as the alteration in permanent winter soil frost for DOC production and soil microbial activity and composition.

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Acknowledgements

Looking back at my time in Umeå since Jan 2007, I have had the opportunity to work in a dynamic research group and on an interesting topic, but have also spent an enjoyable period full of experiences and adventures and come across so many wonderful people.

I would like to express my deepest gratitude to you Hjalmar! Your positive attitude, tact and consideration made this work such a pleasant experience for me.

Mats Öquist, Many thanks for constructive discussions and valuable comments on my manuscripts. Mats Jansson, Thank you for giving me the opportunity to get involved in this PhD project.

Acknowledgments to Swedish Research Council (VR), Oscar and Lili Lamm Foundation, Umeå University (UmU) and the Kempe Foundation who generously provided the financial support for this thesis.

I appreciate the help of Pia Lindell, Anna Nilsson, Viktor Sjäblom, Jennifer Druais, Ida Taberman, Annika Zachrisson, Mark Blackburn, Lukas Mustajärvi, Lenka Kuglerova and Peder Blomkvist who skillfully and devotedly assisted me with the lab and field works. Peder, I value your commitment, exceptional skills and willingness to help.

I am grateful to Ishi Buffam, Kevin Bishop, Johannes Rousk, Erland Bååth, Martin Berggren, Mikaell Ottosson-Löfvenius for fruitful discussions and brilliant comments. Anneli Ågren, Thank you so much for being such a good friend and mentor for me. Du är verkligen "K"anneli som kan allt! Ulrik Ilstedt, Thanks for your patience! Our long discussions on multivariate statistics and design of experiment helped me a lot. Juergen Kreyling, I am looking forward to exploring the frost effects on decomposition in "our" soils! Ryan Sponseller, your contribution with language editing is appreciated.

I have been based at the Dept. of Forest Ecology and Management, SLU for nearly four years. Many thanks to the former and current fellow PhD students and colleagues whom I spent my working days with. Anna, Jakob, Andrés, Katie, Ylva et al., Thank you for being a source of joy and inspiration. You deserve special thanks for enduring my loud expressions of emotion (!) in our office! Åsa, du är så god! Tack för att du tog dig tid att upptäcka saker i min kappa som jag själv inte kunde hitta! Fredrik, lycka till med din fina snabbväxande familj! Rose-Marie, Ida och Björn, tack för vår gemensamma undervisning på "räknestugor" i kemin.

My gratitude to the Bahá'í Community of Umeå who warmly welcomed me and was a source of spiritual inspiration the whole way through.

Mina fina "bonusvänner", Maria, Anna-Carin och Lars, det har varit så trevligt att få lära känna er. Ebba, tack för våra fina lunchträffar genom åren. Hanna, tusen tack för ditt engagemang och att du bryr dig så mycket om mig och alla andra! Ida, du är en så fin vän och resepartner! Som tur kunde jag hitta vägen i Budapest med din hjälp!! Niclas, jag är oerhört glad för att ha fått ta del av din varma, omtanke och tålamod. Tack för att du har varit en källa av trygghet, inspiration och glädje under åren i Umeå!

!مرازگساپس ديدرک مراثن هشيمھ هک یتيامح و قشع مامت رطاخ هب ،مزيزع ردپ و ردام یشاب قفوم هشيمھ هک مراوديما .منونمم تياھيبوخ همھ زا ،مبوخ تسود و ردارب ،بيدا

!

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