Below-ground processes in meadow soil under elevated ozone and carbon dioxide : - Greenhouse gas fluxes, N cycling and microbial communities

This thesis focuses on how elevated CO2 and/or O3 affect the below-ground processes in semi-natural vegetation, with an emphasis on greenhouse gases, N cycling and microbial communities. Meadow mesocosms mimicking lowland hay meadows in Jokioinen, SW Finland, were enclosed in open-top chambers and exposed to ambient and elevated levels of O3 (40-50 ppb) and/or CO2 (+100 ppm) for three consecutive growing season, while chamberless plots were used as chamber controls. Chemical and microbiological analyses as well as laboratory incubations of the mesocosm soils under different treatments were used to study the effects of O3 and/or CO2. Artificially constructed mesocosms were also compared with natural meadows with regards to GHG fluxes and soil characteristics. In addition to research conducted at the ecosystem level (i.e. the mesocosm study), soil microbial communities were also examined in a pot experiment with monocultures of individual species. By comparing mesocosms with similar natural plant assemblage, it was possible to demonstrate that artificial mesocosms simulated natural habitats, even though some differences were found in the CH4 oxidation rate, soil mineral N, and total C and N concentrations in the soil. After three growing seasons of fumigations, the fluxes of N2O, CH4, and CO2 were decreased in the NF+O3 treatment, and the soil NH4+-N and mineral N concentrations were lower in the NF+O3 treatment than in the NF control treatment. The mesocosm soil microbial communities were affected negatively by the NF+O3 treatment, as the total, bacterial, actinobacterial, and fungal PLFA biomasses as well as the fungal:bacterial biomass ratio decreased under elevated O3. In the pot survey, O3 decreased the total, bacterial, actinobacterial, and mycorrhizal PLFA biomasses in the bulk soil and affected the microbial community structure in the rhizosphere of L. pratensis, whereas the bulk soil and rhizosphere of the other monoculture, A. capillaris, remained unaffected by O3. Elevated CO2 caused only minor and insignificant changes in the GHG fluxes, N cycling, and the microbial community structure. In the present study, the below-ground processes were modified after three years of moderate O3 enhancement. A tentative conclusion is that a decrease in N availability may have feedback effects on plant growth and competition and affect the N cycling of the whole meadow ecosystem. Ecosystem level changes occur slowly, and multiplication of the responses might be expected in the long run.

Effects of recreational use and fragmentation on the understorey vegetation and soil microbial communities of urban forests in southern Finland

The impacts of fragmentation and recreational use on the hemiboreal urban forest understorey vegetation and the microbial community of the humus layer (the phospholipid fatty acid (PLFA) pattern, microbial biomass and microbial activity, measured as basal respiration) were examined in the greater Helsinki area, southern Finland. Trampling tolerance of 1) herb-rich OMT, 2) mesic MT, and 3) sub-xeric VT forests (in decreasing order of fertility) was studied by comparing relative understorey vegetation cover (urban/untrampled reference ratio) of the three forest types. The trampling tolerance of forest vegetation increased with the productivity of the site (sub-xeric < mesic < herb-rich). Wear of understorey vegetation correlated positively with the number of residents (i.e., recreational pressure) around the forest patch. An increase of 15000 residents within a radius of 1 km around a forest patch was associated with ca. 30% decrease in the relative understorey vegetation cover. The cover of dwarf shrub Vaccinium myrtillus in particular decreased with increasing levels of wear. The cover of mosses in urban forests was less than half of that in untrampled reference areas. Cover of tree saplings, mainly Sorbus aucuparia, and some resilient herbs was higher than in the reference areas. In small urban forest fragments, broad-leaved trees, grasses and herbs were more abundant and mosses were scarcer than in larger urban forest areas. Thus, due to trampling and edge effects, resilient herb and grass species are replacing sensitive dwarf shrubs, mosses and lichens in urban forests. Differences in the soil microbial community structure were found between paths and untrampled areas and the effects of paths extended more than one meter from the paths. Paths supported approximately 25-30% higher microbial biomass with a transition zone of at least 1 m from the path edge. However, microbial activity per unit of biomass was lower on paths than in untrampled areas. Furthermore, microbial biomass and activity were 30-45% lower at the first 20 m into the forest fragments, due to low moisture content of humus near the edge. The decreased microbial activity detected at forest edges and paths implies decreased litter decomposition rates, and thus, a change in nutrient cycling. Changes in the decomposition and nutrient supply may in turn affect the diversity and function of plant communities in urban forests. Keywords: boreal forest vegetation, edge effects, phospholipid fatty acids, trampling, urban woodlands, wear

Responses of soil microbial communities to clonal variation of Norway spruce

In boreal forests, microorganisms have a pivotal role in nutrient and water supply of trees as well as in litter decomposition and nutrient cycling. This reinforces the link between above-ground and below-ground communities in the context of sustainable productivity of forest ecosystems. In northern boreal forests, the diversity of microbes associated with the trees is high compared to the number of distinct tree species. In this thesis, the aim was to study whether conspecific tree individuals harbour different soil microbes and whether the growth of the trees and the community structure of the associated microbes are connected. The study was performed in a clonal field trial of Norway spruce, which was established in a randomized block design in a clear-cut area. Since out-planting in 1994, the spruce clones showed two-fold growth differences. The fast-growing spruce clones were associated with a more diverse community of ectomycorrhizal fungi than the slow-growing spruce clones. These growth performance groups also differed with respect to other aspects of the associated soil microorganisms: the species composition of ectomycorrhizal fungi, in the amount of extraradical fungal mycelium, in the structure of bacterial community associated with the mycelium, and in the structure of microbial community in the organic layer. The communities of fungi colonizing needle litter of the spruce clones in the field did not differ and the loss of litter mass after two-years decomposition was equal. In vitro, needles of the slow-growing spruce clones were colonized by a more diverse community of endophytic fungi that were shown to be significant needle decomposers. This study showed a relationship between the growth of Norway spruce clones and the community structure of the associated soil microbes. Spatial heterogeneity in soil microbial community was connected with intraspecific variation of trees. The latter may therefore influence soil biodiversity in monospecific forests.

The potential of microbial ecological indicators to guide ecosophisticated management of hydrocarbon-contaminated soils

Soil is an unrenewable natural resource under increasing anthropogenic pressure. One of the main threats to soils, compromising their ability to provide us with the goods and ecosystem services we expect, is pollution. Oil hydrocarbons are the most common soil contaminants, and they disturb not just the biota but also the physicochemical properties of soils. Indigenous soil micro-organisms respond rapidly to changes in the soil ecosystem, and are chronically in direct contact with the hydrophobic pollutants on the soil surfaces. Soil microbial variables could thus serve as an intrinsically relevant indicator of soil quality, to be used in the ecological risk assessment of contaminated and remediated soils. Two contrasting studies were designed to investigate soil microbial ecological responses to hydrocarbons, together with parallel changes in soil physicochemical and ecotoxicological properties. The aim was to identify quantitative or qualitative microbiological variables that would be practicable and broadly applicable for the assessment of the quality and restoration of oil-polluted soil. Soil bacteria commonly react on hydrocarbons as a beneficial substrate, which lead to a positive response in the classical microbiological soil quality indicators; negative impacts were accurately reflected only after severe contamination. Hydrocarbon contaminants become less bioavailable due to weathering processes, and their potentially toxic effects decrease faster than the total concentration. Indigenous hydrocarbon degrader bacteria, naturally present in any terrestrial environment, use specific mechanisms to improve access to the hydrocarbon molecules adsorbed on soil surfaces. Thus when contaminants are unavailable even to the specialised degraders, they should pose no hazard to other biota either. Change in the ratio of hydrocarbon degrader numbers to total microbes was detected to predictably indicate pollutant effects and bioavailability. Also bacterial diversity, a qualitative community characteristic, decreased as a response to hydrocarbons. Stabilisation of community evenness, and community structure that reflected clean reference soil, indicated community recovery. If long-term temporal monitoring is difficult and appropriate clean reference soil unavailable, such comparison could possibly be based on DNA-based community analysis, reflecting past+present, and RNA-based community analysis, showing exclusively present conditions. Microbial ecological indicators cannot replace chemical oil analyses, but they are theoretically relevant and operationally practicable additional tools for ecological risk assessment. As such, they can guide ecologically informed and sustainable ecosophisticated management of oil-contaminated lands.

Bayesian analysis of community dynamics

Elucidating the mechanisms responsible for the patterns of species abundance, diversity, and distribution within and across ecological systems is a fundamental research focus in ecology. Species abundance patterns are shaped in a convoluted way by interplays between inter-/intra-specific interactions, environmental forcing, demographic stochasticity, and dispersal. Comprehensive models and suitable inferential and computational tools for teasing out these different factors are quite limited, even though such tools are critically needed to guide the implementation of management and conservation strategies, the efficacy of which rests on a realistic evaluation of the underlying mechanisms. This is even more so in the prevailing context of concerns over climate change progress and its potential impacts on ecosystems. This thesis utilized the flexible hierarchical Bayesian modelling framework in combination with the computer intensive methods known as Markov chain Monte Carlo, to develop methodologies for identifying and evaluating the factors that control the structure and dynamics of ecological communities. These methodologies were used to analyze data from a range of taxa: macro-moths (Lepidoptera), fish, crustaceans, birds, and rodents. Environmental stochasticity emerged as the most important driver of community dynamics, followed by density dependent regulation; the influence of inter-specific interactions on community-level variances was broadly minor. This thesis contributes to the understanding of the mechanisms underlying the structure and dynamics of ecological communities, by showing directly that environmental fluctuations rather than inter-specific competition dominate the dynamics of several systems. This finding emphasizes the need to better understand how species are affected by the environment and acknowledge species differences in their responses to environmental heterogeneity, if we are to effectively model and predict their dynamics (e.g. for management and conservation purposes). The thesis also proposes a model-based approach to integrating the niche and neutral perspectives on community structure and dynamics, making it possible for the relative importance of each category of factors to be evaluated in light of field data.

Aerobic carbon-cycle related microbial communities in boreal peatlands: responses to water-level drawdown

Boreal peatlands represent a considerable portion of the global carbon (C) pool. Water-level drawdown (WLD) causes peatland drying and induces a vegetation change, which affects the decomposition of soil organic matter and the release of greenhouse gases (CO2 and CH4). The objective of this thesis was to study the microbial communities related to the C cycle and their response to WLD in two boreal peatlands. Both sampling depth and site type had a strong impact on all microbial communities. In general, bacteria dominated the deeper layers of the nutrient-rich fen and the wettest surfaces of the nutrient-poor bog sites, whereas fungi seemed more abundant in the drier surfaces of the bog. WLD clearly affected the microbial communities but the effect was dependent on site type. The fungal and methane-oxidizing bacteria (MOB) community composition changed at all sites but the actinobacterial community response was apparent only in the fen after WLD. Microbial communities became more similar among sites after long-term WLD. Litter quality had a large impact on community composition, whereas the effects of site type and WLD were relatively minor. The decomposition rate of fresh organic matter was influenced slightly by actinobacteria, but not at all by fungi. Field respiration measurements in the northern fen indicated that WLD accelerates the decomposition of soil organic matter. In addition, a correlation between activity and certain fungal sequences indicated that community composition affects the decomposition of older organic matter in deeper peat layers. WLD had a negative impact on CH4 oxidation, especially in the oligotrophic fen. Fungal sequences were matched to taxa capable of utilizing a broad range of substrates. Most of the actinobacterial sequences could not be matched to characterized taxa in reference databases. This thesis represents the first investigation of microbial communities and their response to WLD among a variety of boreal peatland habitats. The results indicate that microbial community responses to WLD are complex but dependent on peatland type, litter quality, depth, and variable among microbes.

Mycorrhizal fungi and nitrogen dynamics in drained peatland

Fungi have a fundamental role in carbon and nutrient transformations in the acids soils of boreal regions, such as peatlands, where high amounts of carbon (C) and nutrients are stored in peat, the pH is relatively low and the nutrient uptake of trees is highly dependent on mycorrhizae. In this thesis, the aim was to examine nitrogen (N) transformations and the availability of dissolved N compounds in forestry-drained peatlands, to compare the fungal community biomass and structure at various peat N levels, to investigate the growth of ectomycorrhizal fungi with variable P and K availability and to assess how the ectomycorrhizal fungi (ECM) affect N transformations. Both field and laboratory experiments were carried out. The peat N concentration did not affect the soil fungal community structure within a site. Phosphorus (P) and potassium (K) deficiency of the trees as well as the degree of decomposition and dissolved organic nitrogen (DON) concentration of the peat were shown to affect the fungal community structure and biomass of ECMs, highlighting the complexity of the below ground system on drained peatlands. The biomass of extrametrical mycorrhizal mycelia (EMM) was enhanced by P and/or K deficiency of the trees, and ECM biomass in the roots was increased by P deficiency. Thus, PK deficiency in drained peatlands may increase the allocation of C by the tree to ECMs. It was also observed that fungi can alter N mineralization processes in the rhizosphere but variously depending on fungal species and fertility level of peat. Gross N mineralization did not vary but the net N mineralization rate significantly increased along the N gradient in both field and laboratory experiments. Gross N immobilization also significantly increased when the peat N concentration increased. Nitrification was hardly detectable in either field or laboratory experiments. During the growing season, dissolved inorganic N (DIN) fluctuated much more than the relatively stable DON. Special methodological challenges associated with sampling and analysis in microbial studies on peatlands are discussed.

Microbial communities in Pb contaminated boreal forest soil

Lead contamination in the environment is of particular concern, as it is a known toxin. Until recently, however, much less attention has been given to the local contamination caused by activities at shooting ranges compared to large-scale industrial contamination. In Finland, more than 500 tons of Pb is produced each year for shotgun ammunition. The contaminant threatens various organisms, ground water and the health of human populations. However, the forest at shooting ranges usually shows no visible sign of stress compared to nearby clean environments. The aboveground biota normally reflects the belowground ecosystem. Thus, the soil microbial communities appear to bear strong resistance to contamination, despite the influence of lead. The studies forming this thesis investigated a shooting range site at Hälvälä in Southern Finland, which is heavily contaminated by lead pellets. Previously it was experimentally shown that the growth of grasses and degradation of litter are retarded. Measurements of acute toxicity of the contaminated soil or soil extracts gave conflicting results, as enchytraeid worms used as toxicity reporters were strongly affected, while reporter bacteria showed no or very minor decreases in viability. Measurements using sensitive inducible luminescent reporter bacteria suggested that the bioavailability of lead in the soil is indeed low, and this notion was supported by the very low water extractability of the lead. Nevertheless, the frequency of lead-resistant cultivable bacteria was elevated based on the isolation of cultivable strains. The bacterial and fungal diversity in heavily lead contaminated shooting sectors were compared with those of pristine sections of the shooting range area. The bacterial 16S rRNA gene and fungal ITS rRNA gene were amplified, cloned and sequenced using total DNA extracted from the soil humus layer as the template. Altogether, 917 sequenced bacterial clones and 649 sequenced fungal clones revealed a high soil microbial diversity. No effect of lead contamination was found on bacterial richness or diversity, while fungal richness and diversity significantly differed between lead contaminated and clean control areas. However, even in the case of fungi, genera that were deemed sensitive were not totally absent from the contaminated area: only their relative frequency was significantly reduced. Some operational taxonomic units (OTUs) assigned to Basidiomycota were clearly affected, and were much rarer in the lead contaminated areas. The studies of this thesis surveyed EcM sporocarps, analyzed morphotyped EcM root tips by direct sequencing, and 454-pyrosequenced fungal communities in in-growth bags. A total of 32 EcM fungi that formed conspicuous sporocarps, 27 EcM fungal OTUs from 294 root tips, and 116 EcM fungal OTUs from a total of 8 194 ITS2 454 sequences were recorded. The ordination analyses by non-parametric multidimensional scaling (NMS) indicated that Pb enrichment induced a shift in the EcM community composition. This was visible as indicative trends in the sporocarp and root tip datasets, but explicitly clear in the communities observed in the in-growth bags. The compositional shift in the EcM community was mainly attributable to an increase in the frequencies of OTUs assigned to the genus Thelephora, and to a decrease in the OTUs assigned to Pseudotomentella, Suillus and Tylospora in Pb-contaminated areas when compared to the control. The enrichment of Thelephora in contaminated areas was also observed when examining the total fungal communities in soil using DNA cloning and sequencing technology. While the compositional shifts are clear, their functional consequences for the dominant trees or soil ecosystem remain undetermined. The results indicate that at the Hälvälä shooting range, lead influences the fungal communities but not the bacterial communities. The forest ecosystem shows apparent functional redundancy, since no significant effects were seen on forest trees. Recently, by means of 454 pyrosequencing , the amount of sequences in a single analysis run can be up to one million. It has been applied in microbial ecology studies to characterize microbial communities. The handling of sequence data with traditional programs is becoming difficult and exceedingly time consuming, and novel tools are needed to handle the vast amounts of data being generated. The field of microbial ecology has recently benefited from the availability of a number of tools for describing and comparing microbial communities using robust statistical methods. However, although these programs provide methods for rapid calculation, it has become necessary to make them more amenable to larger datasets and numbers of samples from pyrosequencing. As part of this thesis, a new program was developed, MuSSA (Multi-Sample Sequence Analyser), to handle sequence data from novel high-throughput sequencing approaches in microbial community analyses. The greatest advantage of the program is that large volumes of sequence data can be manipulated, and general OTU series with a frequency value can be calculated among a large number of samples.

Microbial diversity in the municipal composting process and development of detection methods

Composting refers to aerobic degradation of organic material and is one of the main waste treatment methods used in Finland for treating separated organic waste. The composting process allows converting organic waste to a humus-like end product which can be used to increase the organic matter in agricultural soils, in gardening, or in landscaping. Microbes play a key role as degraders during the composting-process, and the microbiology of composting has been studied for decades, but there are still open questions regarding the microbiota in industrial composting processes. It is known that with the traditional, culturing-based methods only a small fraction, below 1%, of the species in a sample is normally detected. In recent years an immense diversity of bacteria, fungi and archaea has been found to occupy many different environments. Therefore the methods of characterising microbes constantly need to be developed further. In this thesis the presence of fungi and bacteria in full-scale and pilot-scale composting processes was characterised with cloning and sequencing. Several clone libraries were constructed and altogether nearly 6000 clones were sequenced. The microbial communities detected in this study were found to differ from the compost microbes observed in previous research with cultivation based methods or with molecular methods from processes of smaller scale, although there were similarities as well. The bacterial diversity was high. Based on the non-parametric coverage estimations, the number of bacterial operational taxonomic units (OTU) in certain stages of composting was over 500. Sequences similar to Lactobacillus and Acetobacteria were frequently detected in the early stages of drum composting. In tunnel stages of composting the bacterial community comprised of Bacillus, Thermoactinomyces, Actinobacteria and Lactobacillus. The fungal diversity was found to be high and phylotypes similar to yeasts were abundantly found in the full-scale drum and tunnel processes. In addition to phylotypes similar to Candida, Pichia and Geotrichum moulds from genus Thermomyces and Penicillium were observed in tunnel stages of composting. Zygomycetes were detected in the pilot-scale composting processes and in the compost piles. In some of the samples there were a few abundant phylotypes present in the clone libraries that masked the rare ones. The rare phylotypes were of interest and a method for collecting them from clone libraries for sequencing was developed. With negative selection of the abundant phylotyps the rare ones were picked from the clone libraries. Thus 41% of the clones in the studied clone libraries were sequenced. Since microbes play a central role in composting and in many other biotechnological processes, rapid methods for characterization of microbial diversity would be of value, both scientifically and commercially. Current methods, however, lack sensitivity and specificity and are therefore under development. Microarrays have been used in microbial ecology for a decade to study the presence or absence of certain microbes of interest in a multiplex manner. The sequence database collected in this thesis was used as basis for probe design and microarray development. The enzyme assisted detection method, ligation-detection-reaction (LDR) based microarray, was adapted for species-level detection of microbes characteristic of each stage of the composting process. With the use of a specially designed control probe it was established that a species specific probe can detect target DNA representing as little as 0.04% of total DNA in a sample. The developed microarray can be used to monitor composting processes or the hygienisation of the compost end product. A large compost microbe sequence dataset was collected and analysed in this thesis. The results provide valuable information on microbial community composition during industrial scale composting processes. The microarray method was developed based on the sequence database collected in this study. The method can be utilised in following the fate of interesting microbes during composting process in an extremely sensitive and specific manner. The platform for the microarray is universal and the method can easily be adapted for studying microbes from environments other than compost.

Plasmids and aromatic degradation in Sphingomonas for bioremediation : Aromatic ring cleavage genes in soil and rhizosphere

Microbial degradation pathways play a key role in the detoxification and the mineralization of polyaromatic hydrocarbons (PAHs), which are widespread pollutants in soil and constituents of petroleum hydrocarbons. In microbiology the aromatic degradation pathways are traditionally studied from single bacterial strains with capacity to degrade certain pollutant. In soil the degradation of aromatics is performed by a diverse community of micro-organisms. The aim of this thesis was to study biodegradation on different levels starting from a versatile aromatic degrader Sphingobium sp. HV3 and its megaplasmid, extending to revelation of diversity of key catabolic enzymes in the environment and finally studying birch rhizoremediation in PAH-polluted soil. To understand biodegradation of aromatics on bacterial species level, the aromatic degradation capacity of Sphingobium sp. HV3 and the role of the plasmid pSKY4, was studied. Toluene, m-xylene, biphenyl, fluorene, phenanthrene were detected as carbon and energy sources of the HV3 strain. Tn5 transposon mutagenesis linked the degradation capacity of toluene, m-xylene, biphenyl and naphthalene to the pSKY4 plasmid and qPCR expression analysis showed that plasmid extradiol dioxygenases genes (bphC and xylE) are inducted by phenanthrene, m-xylene and biphenyl whereas the 2,4-dichlorophenoxyacetic acid herbicide induced the chlorocatechol 1,2-dioxygenase gene (tfdC) from the ortho-pathway. A method to study upper meta-pathway extradiol dioxygenase gene diversity in soil was developed. The extradiol dioxygenases catalyse cleavage of the aromatic ring between a hydroxylated carbon and an adjacent non-hydroxylated carbon (meta-cleavage). A high diversity of extradiol dioxygenases were detected from polluted soils. The detected extradiol dioxygenases showed sequence similarity to known catabolic genes of Alpha-, Beta-, and Gammaproteobacteria. Five groups of extradiol dioxygenases contained sequences with no close homologues in the database, representing novel genes. In rhizoremediation experiment with birch (Betula pendula) treatment specific changes of extradiol dioxygenase communities were shown. PAH pollution changed the bulk soil extradiol dioxygenase community structure and birch rhizosphere contained a more diverse extradiol dioxygenase community than the bulk soil showing a rhizosphere effect. The degradation of pyrene in soil was enhanced with birch seedlings compared to soil without birch. The complete 280,923 kb nucleotide sequence of pSKY4 plasmid was determined. The open reading frames of pSKY4 were divided into putative conjugative transfer, aromatic degradation, replication/maintaining and transposition/integration function-encoding proteins. Aromatic degradation orfs shared high similarity to corresponding genes in pNL1, a plasmid from the deep subsurface strain Novosphingobium aromaticivorans F199. The plasmid backbones were considerably more divergent with lower similarity, which suggests that the aromatic pathway has functioned as a plasmid independent mobile genetic element. The functional diversity of microbial communities in soil is still largely unknown. Several novel clusters of extradiol dioxygenases representing catabolic bacteria, whose function, biodegradation pathways and phylogenetic position is not known were amplified with single primer pair from polluted soils. These extradiol dioxygenase communities were shown to change upon PAH pollution, which indicates that their hosts function in PAH biodegradation in soil. Although the degradation pathways of specific bacterial species are substantially better depicted than pathways in situ, the evolution of degradation pathways for the xenobiotic compounds is largely unknown. The pSKY4 plasmid contains aromatic degradation genes in putative mobile genetic element causing flexibility/instability to the pathway. The localisation of the aromatic biodegradation pathway in mobile genetic elements suggests that gene transfer and rearrangements are a competetive advantage for Sphingomonas bacteria in the environment.

Carbon dynamics in peatlands under changing hydrology : Effects of water level drawdown on litter quality, microbial enzyme activities and litter decomposition rates

Pristine peatlands are carbon (C) accumulating wetland ecosystems sustained by a high water level (WL) and consequent anoxia that slows down decomposition. Persistent WL drawdown as a response to climate and/or land-use change directly affects decomposition: increased oxygenation stimulates decomposition of the old C (peat) sequestered under prior anoxic conditions. Responses of the new C (plant litter) in terms of quality, production and decomposability, and the consequences for the whole C cycle of peatlands are not fully understood. WL drawdown induces changes in plant community resulting in shift in dominance from Sphagnum and graminoids to shrubs and trees. There is increasing evidence that the indirect effects of WL drawdown via the changes in plant communities will have more impact on the ecosystem C cycling than any direct effects. The aim of this study is to disentangle the direct and indirect effects of WL drawdown on the new C by measuring the relative importance of 1) environmental parameters (WL depth, temperature, soil chemistry) and 2) plant community composition on litter production, microbial activity, litter decomposition rates and, consequently, on the C accumulation. This information is crucial for modelling C cycle under changing climate and/or land-use. The effects of WL drawdown were tested in a large-scale experiment with manipulated WL at two time scales and three nutrient regimes. Furthermore, the effect of climate on litter decomposability was tested along a north-south gradient. Additionally, a novel method for estimating litter chemical quality and decomposability was explored by combining Near infrared spectroscopy with multivariate modelling. WL drawdown had direct effects on litter quality, microbial community composition and activity and litter decomposition rates. However, the direct effects of WL drawdown were overruled by the indirect effects via changes in litter type composition and production. Short-term (years) responses to WL drawdown were small. In long-term (decades), dramatically increased litter inputs resulted in large accumulation of organic matter in spite of increased decomposition rates. Further, the quality of the accumulated matter greatly changed from that accumulated in pristine conditions. The response of a peatland ecosystem to persistent WL drawdown was more pronounced at sites with more nutrients. The study demonstrates that the shift in vegetation composition as a response to climate and/or land-use change is the main factor affecting peatland ecosystem C cycle and thus dynamic vegetation is a necessity in any models applied for estimating responses of C fluxes to changes in the environment. The time scale for vegetation changes caused by hydrological changes needs to extend to decades. This study provides grouping of litter types (plant species and part) into functional types based on their chemical quality and/or decomposability that the models could utilize. Further, the results clearly show a drop in soil temperature as a response to WL drawdown when an initially open peatland converts into a forest ecosystem, which has not yet been considered in the existing models.

Fast- and drift-ice communities in the Bothnian Bay and the impact of UVA radiation on the Baltic Sea ice ecology

The aim of this thesis was to study ecology of Baltic Sea ice from two perspectives. In the first two studies, sea-ice ecology from riverine-influenced fast ice to drift ice in the Bothnian Bay was investigated, whereas the last two studies focus on the sensitivity of sea-ice bacteria and algae to UVA examined in situ. The seasonal sea ice cover is one of the main characteristics of the Baltic Sea, and despite the brackish parental water, the ice structure is similar to polar ice with saline brine inclusions, the sea ice habitat. The decreasing seawater salinity from the northern Baltic Sea to the Bothnian Bay translates to decreasing brine volumes along the gradient, governing the size and community structure of the food webs in ice. However, the drift and fast ice in the Bothnian Bay may differ greatly in this sense, as drift ice may have been formed at more southern locations. Rafting and the formation of snow ice are common processes in the ice field of the Bothnian Bay. As evidenced in this thesis, rafting altered the vertical distribution of organisms and snow-ice formation provided habitable space in the better-illuminated, nitrogen-rich surface layer. The divergence between fast and drift ice became apparent at the more advanced stages, and chlorophyte biomass decreased from fast to drift ice, while the opposite held true for protozoan and metazoan biomass. The brine volumes affected the communities somewhat, and a higher percentage of flagellate species was generally linked to lower brine volumes, whereas chain-forming diatoms were mostly concentrated in layers with larger brine volumes. These results add to knowledge of the ecological significance of the ice cover lasting up to 7 months per year in this area. Sea-ice food webs are generally light-limited, but while increasing light irradiances typically enhance the primary production and further, the secondary production in sea ice, any increase in solar radiation also includes an increase in harmful UVA radiation. The Baltic Sea ice microbial communities were clearly sensitive to UVA and the responses were strongly linked to the earlier light history, as well as to the solar irradiances they were exposed to. The increased biomass of chlorophytes and pennate diatoms, when UVA was excluded, indicates that their normally minor contribution to the biomass in the upper layers of sea ice might be partly dictated by UVA. The effects of UVA on bacterial production in Baltic Sea ice mostly followed the responses in algal growth, but occasionally the exposure to UVA even enhanced the bacterial production. The dominant bacterial class, Flavobacteria, seemed to be UVA-tolerant, whereas all the Alpha-, Beta- and Gammaproteobacteria present in the surface layer showed UVA sensitivity. These results indicate that changes in the light field of ice may alter the community structure and affect the functioning of ice food webs, and are of importance when the effects of thinning of the ice cover are assessed.

Microbiological characterisation of soils : Evaluation of some critical steps in data collection and experimental design

The study of soil microbiota and their activities is central to the understanding of many ecosystem processes such as decomposition and nutrient cycling. The collection of microbiological data from soils generally involves several sequential steps of sampling, pretreatment and laboratory measurements. The reliability of results is dependent on reliable methods in every step. The aim of this thesis was to critically evaluate some central methods and procedures used in soil microbiological studies in order to increase our understanding of the factors that affect the measurement results and to provide guidance and new approaches for the design of experiments. The thesis focuses on four major themes: 1) soil microbiological heterogeneity and sampling, 2) storage of soil samples, 3) DNA extraction from soil, and 4) quantification of specific microbial groups by the most-probable-number (MPN) procedure. Soil heterogeneity and sampling are discussed as a single theme because knowledge on spatial (horizontal and vertical) and temporal variation is crucial when designing sampling procedures. Comparison of adjacent forest, meadow and cropped field plots showed that land use has a strong impact on the degree of horizontal variation of soil enzyme activities and bacterial community structure. However, regardless of the land use, the variation of microbiological characteristics appeared not to have predictable spatial structure at 0.5-10 m. Temporal and soil depth-related patterns were studied in relation to plant growth in cropped soil. The results showed that most enzyme activities and microbial biomass have a clear decreasing trend in the top 40 cm soil profile and a temporal pattern during the growing season. A new procedure for sampling of soil microbiological characteristics based on stratified sampling and pre-characterisation of samples was developed. A practical example demonstrated the potential of the new procedure to reduce the analysis efforts involved in laborious microbiological measurements without loss of precision. The investigation of storage of soil samples revealed that freezing (-20 °C) of small sample aliquots retains the activity of hydrolytic enzymes and the structure of the bacterial community in different soil matrices relatively well whereas air-drying cannot be recommended as a storage method for soil microbiological properties due to large reductions in activity. Freezing below -70 °C was the preferred method of storage for samples with high organic matter content. Comparison of different direct DNA extraction methods showed that the cell lysis treatment has a strong impact on the molecular size of DNA obtained and on the bacterial community structure detected. An improved MPN method for the enumeration of soil naphthalene degraders was introduced as an alternative to more complex MPN protocols or the DNA-based quantification approach. The main advantage of the new method is the simple protocol and the possibility to analyse a large number of samples and replicates simultaneously.

Microbial identification by detection of ligation probes on DNA microarray

Microbes in natural and artificial environments as well as in the human body are a key part of the functional properties of these complex systems. The presence or absence of certain microbial taxa is a correlate of functional status like risk of disease or course of metabolic processes of a microbial community. As microbes are highly diverse and mostly notcultivable, molecular markers like gene sequences are a potential basis for detection and identification of key types. The goal of this thesis was to study molecular methods for identification of microbial DNA in order to develop a tool for analysis of environmental and clinical DNA samples. Particular emphasis was placed on specificity of detection which is a major challenge when analyzing complex microbial communities. The approach taken in this study was the application and optimization of enzymatic ligation of DNA probes coupled with microarray read-out for high-throughput microbial profiling. The results show that fungal phylotypes and human papillomavirus genotypes could be accurately identified from pools of PCR amplicons generated from purified sample DNA. Approximately 1 ng/μl of sample DNA was needed for representative PCR amplification as measured by comparisons between clone sequencing and microarray. A minimum of 0,25 amol/μl of PCR amplicons was detectable from amongst 5 ng/μl of background DNA, suggesting that the detection limit of the test comprising of ligation reaction followed by microarray read-out was approximately 0,04%. Detection from sample DNA directly was shown to be feasible with probes forming a circular molecule upon ligation followed by PCR amplification of the probe. In this approach, the minimum detectable relative amount of target genome was found to be 1% of all genomes in the sample as estimated from 454 deep sequencing results. Signal-to-noise of contact printed microarrays could be improved by using an internal microarray hybridization control oligonucleotide probe together with a computational algorithm. The algorithm was based on identification of a bias in the microarray data and correction of the bias as shown by simulated and real data. The results further suggest semiquantitative detection to be possible by ligation detection, allowing estimation of target abundance in a sample. However, in practise, comprehensive sequence information of full length rRNA genes is needed to support probe design with complex samples. This study shows that DNA microarray has the potential for an accurate microbial diagnostic platform to take advantage of increasing sequence data and to replace traditional, less efficient methods that still dominate routine testing in laboratories. The data suggests that ligation reaction based microarray assay can be optimized to a degree that allows good signal-tonoise and semiquantitative detection.