Foliar potassium concentrations of bilberry, bog bilberry and downy birch as indicators of potassium nutrition of Scots pine on a drained peatland

Abstract

Improvements in the Assessment of Bacterial Viability and Killing

It is axiomatic that our planet is extensively inhabited by diverse micro-organisms such as bacteria, yet the absolute diversity of different bacterial species is widely held to be unknown. Different bacteria can be found from the depths of the oceans to the top of the mountains; even the air is more or less colonized by bacteria. Most bacteria are either harmless or even advantageous to human beings but there are also bacteria, which can cause severe infectious diseases or spoil the supplies intended for human consumption. Therefore, it is vitally important not only to be able to detect and enumerate bacteria but also to assess their viability and possible harmfulness. Whilst the growth of bacteria is remarkably fast under optimum conditions and easy to detect by cultural methods, most bacteria are believed to lie in stationary phase of growth in which the actual growth is ceased and thus bacteria may simply be undetectable by cultural techniques. Additionally, several injurious factors such as low and high temperature or deficiency of nutrients can turn bacteria into a viable but non-culturable state (VBNC) that cannot be detected by cultural methods. Thereby, various noncultural techniques developed for the assessment of bacterial viability and killing have widely been exploited in modern microbiology. However, only a few methods are suitable for kinetic measurements, which enable the real-time detection of bacterial growth and viability. The present study describes alternative methods for measuring bacterial viability and killing as well as detecting the effects of various antimicrobial agents on bacteria on a real-time basis. The suitability of bacterial (lux) and beetle (luc) luciferases as well as green fluorescent protein (GFP) to act as a marker of bacterial viability and cell growth was tested. In particular, a multiparameter microplate assay based on GFP-luciferase combination as well as a flow cytometric measurement based on GFP-PI combination were developed to perform divergent viability analyses. The results obtained suggest that the antimicrobial activities of various drugs against bacteria could be successfully measured using both of these methods. Specifically, the data reliability of flow cytometric viability analysis was notably improved as GFP was utilized in the assay. A fluoro-luminometric microplate assay enabled kinetic measurements, which significantly improved and accelerated the assessment of bacterial viability compared to more conventional viability assays such as plate counting. Moreover, the multiparameter assay made simultaneous detection of GFP fluorescence and luciferase bioluminescence possible and provided extensive information about multiple cellular parameters in single assay, thereby increasing the accuracy of the assessment of the kinetics of antimicrobial activities on target bacteria.

Inactive endosialidase-based detection of bacterial and oncofetal polysialic acid

Polysialic acid is a carbohydrate polymer which consist of N-acetylneuraminic acid units joined by alpha2,8-linkages. It is developmentally regulated and has an important role during normal neuronal development. In adults, it participates in complex neurological processes, such as memory, neural plasticity, tumor cell growth and metastasis. Polysialic acid also constitutes the capsule of some meningitis and sepsis-causing bacteria, such as Escherichia coli K1, group B meningococci, Mannheimia haemolytica A2 and Moraxella nonliquefaciens. Polysialic acid is poorly immunogenic; therefore high affinity antibodies against it are difficult to prepare, thus specific and fast detection methods are needed. Endosialidase is an enzyme derived from the E. coli K1 bacteriophage, which specifically recognizes and degrades polysialic acid. In this study, a novel detection method for polysialic acid was developed based on a fusion protein of inactive endosialidase and the green fluorescent protein. It utilizes the ability of the mutant, inactive endosialidase to bind but not cleave polysialic acid. Sequencing of the endosialidase gene revealed that amino acid substitutions near the active site of the enzyme differentiate the active and inactive forms of the enzyme. The fusion protein was applied for the detection of polysialic acid in bacteria and neuroblastoma. The results indicate that the fusion protein is a fast, sensitive and specific reagent for the detection of polysialic acid. The use of an inactive enzyme as a specific molecular tool for the detection of its substrate represents an approach which could potentially find wide applicability in the specific detection of diverse macromolecules.

Testate amoebae (thecamoebians) as indicators of aquatic mine impact

The environmental impacts of a single mine often remain local, but acidic and metal-rich acid mine drainage (AMD) from the waste materials may pose a serious threat to adjacent surface waters and their ecosystems. Testate amoebae (thecamoebian) analysis was used together with lake sediment geochemistry to study and evaluate the ecological effects of sulphidic metal mines on aquatic environments. Three different mines were included in the study: Luikonlahti Cu-mine in Kaavi, eastern Finland, Haveri Cu-Au mine in Ylöjärvi, southern Finland and Pyhäsalmi Zn-Cu-S mine in Pyhäjärvi, central Finland. Luikonlahti and Haveri are closed mines, but Pyhäsalmi is still operating. The sampling strategy was case specific, and planned to provide a representative sediment sample series to define natural background conditions, to detect spatial and temporal variations in mine impacts, to evaluate the possible recovery after the peak contamination, and to distinguish the effects of other environmental factors from the mining impacts. In the Haveri case, diatom analyses were performed alongside thecamoebian analysis to evaluate the similarities and differences between the two proxies. The results of the analyses were investigated with multivariate methods (direct and indirect ordinations, diversity and distance measure indices). Finally, the results of each case study were harmonized, pooled, and jointly analyzed to summarize the results for this dissertation. Geochemical results showed broadly similar temporal patterns in each case. Concentrations of ions in the pre-disturbance samples defined the natural baseline against which other results were compared. The beginning of the mining activities had only minor impacts on sediment geochemistry, mainly appearing as an increased clastic input into the lakes at Haveri and Pyhäsalmi. The active mining phase was followed by the metallic contamination and, subsequently, by the most recent change towards decreased but still elevated metal concentrations in the sediments. Because of the delay in the oxidation of waste material and formation of AMD, the most intense, but transient metal contamination phase occurred in the post-mining period at Luikonlahti and Haveri. At Pyhäsalmi, the highest metal contamination preceded effluent mitigation actions. Spatial gradients were observed besides the temporal evolution in both the pre-disturbance and mine-impacted samples from Luikonlahti and Pyhäsalmi. The geochemical gradients varied with distance from the main source of contaminants (dispersion and dilution) and with water depth (redox and pH). The spatial extent of the highest metal contamination associated with these mines remained rather limited. At Haveri, the metallic impact was widespread, with the upstream site in another lake basin found to be contaminated. Changes in thecamoebian assemblages corresponded well with the geochemical results. Despite some differences, the general features and ecological responses of the faunal assemblages were rather similar in each lake. Constantly abundant strains of Difflugia oblonga, Difflugia protaeiformis and centropyxids formed the core of these assemblages. Increasing proportions of Cucurbitella tricuspis towards the surface samples were found in all of the cases. The results affirmed the indicator value of some already known indicator forms, but such as C. tricuspis and higher nutrient levels, but also elicited possible new ones such as D. oblonga ‘spinosa’ and clayey substrate, high conductivity and/or alkalinity, D. protaeiformis ‘multicornis’ and pH, water hardness and the amount of clastic material and Centropyxis constricta ‘aerophila’ and high metal and S concentrations. In each case, eutrophication appeared to be the most important environmental factor, masking the effects of other variables. Faunal responses to high metal inputs in sediments remained minor, but were nevertheless detectable. Besides the trophic state of the lake, numerical methods suggested overall geochemical conditions (pH, redox) to be the most important factor at Luikonlahti, whereas the Haveri results showed the clearest connection between metals and amoebae. At Pyhäsalmi, the strongest relationships were found between Ca- and S-rich present loading, redox conditions and substrate composition. Sediment geochemistry and testate amoeba analysis proved to be a suitable combination of methods to detect and describe the aquatic mine impacts in each specific case, to evaluate recovery and to differentiate between the effects of different anthropogenic and natural environmental factors. It was also suggested that aquatic mine impacts can be significantly mitigated by careful design and after-care of the waste facilities, especially by reducing and preventing AMD. The case-specific approach is nevertheless necessary because of the unique characteristics of each mine and variations in the environmental background conditions.