DNA-based detection and characterisation of strictly anaerobic beer-spoilage bacteria

Megasphaera cerevisiae, Pectinatus cerevisiiphilus, Pectinatus frisingensis, Selenomonas lacticifex, Zymophilus paucivorans and Zymophilus raffinosivorans are strictly anaerobic Gram-stain-negative bacteria that are able to spoil beer by producing off-flavours and turbidity. They have only been isolated from the beer production chain. The species are phylogenetically affiliated to the Sporomusa sub-branch in the class "Clostridia". Routine cultivation methods for detection of strictly anaerobic bacteria in breweries are time-consuming and do not allow species identification. The main aim of this study was to utilise DNA-based techniques in order to improve detection and identification of the Sporomusa sub-branch beer-spoilage bacteria and to increase understanding of their biodiversity, evolution and natural sources. Practical PCR-based assays were developed for monitoring of M. cerevisiae, Pectinatus species and the group of Sporomusa sub-branch beer spoilers throughout the beer production process. The developed assays reliably differentiated the target bacteria from other brewery-related microbes. The contaminant detection in process samples (10 1,000 cfu/ml) could be accomplished in 2 8 h. Low levels of viable cells in finished beer (≤10 cfu/100 ml) were usually detected after 1 3 d culture enrichment. Time saving compared to cultivation methods was up to 6 d. Based on a polyphasic approach, this study revealed the existence of three new anaerobic spoilage species in the beer production chain, i.e. Megasphaera paucivorans, Megasphaera sueciensis and Pectinatus haikarae. The description of these species enabled establishment of phenotypic and DNA-based methods for their detection and identification. The 16S rRNA gene based phylogenetic analysis of the Sporomusa sub-branch showed that the genus Selenomonas originates from several ancestors and will require reclassification. Moreover, Z. paucivorans and Z. raffinosivorans were found to be in fact members of the genus Propionispira. This relationship implies that they were carried to breweries along with plant material. The brewery-related Megasphaera species formed a distinct sub-group that did not include any sequences from other sources, suggesting that M. cerevisiae, M. paucivorans and M. sueciensis may be uniquely adapted to the brewery ecosystem. M. cerevisiae was also shown to exhibit remarkable resistance against many brewery-related stress conditions. This may partly explain why it is a brewery contaminant. This study showed that DNA-based techniques provide useful tools for obtaining more rapid and specific information about the presence and identity of the strictly anaerobic spoilage bacteria in the beer production chain than is possible using cultivation methods. This should ensure financial benefits to the industry and better product quality to customers. In addition, DNA-based analyses provided new insight into the biodiversity as well as natural sources and relations of the Sporomusa sub-branch bacteria. The data can be exploited for taxonomic classification of these bacteria and for surveillance and control of contaminations.

Global and EU governance for sustainable forest management with special reference to capacity building in Ethiopia and Southern Sudan

The purpose of this study was to produce information on and practical recommendations for informed decision-making on and capacity building for sustainable forest management (SFM) and good forest governance. This was done within the overall global framework for sustainable development with special emphasis on the EU and African frameworks and on Southern Sudan and Ethiopia in particular. The case studies on Southern Sudan and Ethiopia focused on local, national and regional issues. Moreover, this study attempted to provide both theoretical and practical new insight. The aim was to build an overall theoretical framework and to study its key contents and main implications for SFM and good forest governance at all administration levels, for providing new tools for capacity building in natural resources management. The theoretical framework and research approach were based on the original research problem and the general and specific aims of the study. The key elements of the framework encompass sustainable development, global and EU governance, sustainable forest management (SFM), good forest governance, as well as international and EU law. The selected research approach comprised matrix-based assessment of international, regional (EU and Africa) and national (Southern Sudan and Ethiopia) policy and legal documents. The specific case study on Southern Sudan also involved interviews and group discussions with local community members and government officials. As a whole, this study attempted to link the global, regional, national and local levels in forest-sector development and especially to analyse how the international policy development in environmental and forestry issues is reflected in field-level progress towards SFM and good forest governance, for the specific cases of Southern Sudan and Ethiopia. The results on Southern Sudan focused on the existing situation and perceived needs in capacity building for SFM and good forest governance at all administration levels. Specifically, the results of the case study on Southern Sudan presented the current situation in selected villages in the northern parts of Renk County in Upper Nile State, and the implications of Multilateral Environmental Agreements (MEAs) and of the new forest policy framework for capacity building actions. The results on Ethiopia focused on training, extension, research, education and new curriculum development within higher education institutions and particularly at the Wondo Genet College of Forestry and Natural Resources (WGCF-NR), which administratively lies under Hawassa University. The results suggest that, for both cases studies, informed decision-making on and capacity building for SFM and good forest governance require comprehensive, long-term, cross-sectoral, coherent and consistent approaches within the dynamic and evolving overall global framework, including its multiple inter-linked levels. The specific priority development and focus areas comprised the establishment of SFM and good forest governance in accordance with the overall sustainable development priorities and with more focus on the international trade in forest products that are derived from sustainable and legal sources with an emphasis on effective forest law enforcement and governance at all levels. In Upper Nile State in Southern Sudan there were positive development signals such as the will of the local people to plant more multipurpose trees on farmlands and range lands as well as the recognition of the importance of forests and trees for sustainable rural development where food security is a key element. In addition, it was evident that the local communities studied in Southern Sudan also wanted to establish good governance systems through partnerships with all actors and through increased local responsibilities. The results also suggest that the implementation of MEAs at the local level in Southern Sudan requires mutually supportive and coherent approaches within the agreements as well as significantly more resources and financial and technical assistance for capacity building, training and extension. Finally, the findings confirm the importance of full utilization of the existing local governance and management systems and their traditional and customary knowledge and practices, and of new development partnerships with full participation of all stakeholders. The planned new forest law for Southern Sudan, based on an already existing new forest policy, is expected to recognize the roles of local-level actors, and it would thus obviously facilitate the achieving of sustainable forest management.

Expression of lactate transporters MCT1, MCT2, MCT4 and the ancillary protein CD147 in horse muscle and red blood cells

Monocarboxylate transporters (MCTs) transport lactate and protons across cell membranes. During intense exercise, lactate and protons accumulate in the exercising muscle and are transported to the plasma. In the horse, MCTs are responsible for the majority of lactate and proton removal from exercising muscle, and are therefore also the main mechanism to hinder the decline in pH in muscle cells. Two isoforms, MCT1 and MCT4, which need an ancillary protein CD147, are expressed in equine muscle. In the horse, as in other species, MCT1 is predominantly expressed in oxidative fibres, where its likely role is to transport lactate into the fibre to be used as a fuel at rest and during light work, and to remove lactate during intensive exercise when anaerobic energy production is needed. The expression of CD147 follows the fibre type distribution of MCT1. These proteins were detected in both the cytoplasm and sarcolemma of muscle cells in the horse breeds studied: Standardbred and Coldblood trotters. In humans, training increases the expression of both MCT1 and MCT4. In this study, the proportion of oxidative fibres in the muscle of Norwegian-Swedish Coldblood trotters increased with training. Simultaneously, the expression of MCT1 and CD147, measured immunohistochemically, seemed to increase more in the cytoplasm of oxidative fibres than in the fast fibre type IIB. Horse MCT4 antibody failed to work in immunohistochemistry. In the future, a quantitative method should be introduced to examine the effect of training on muscle MCT expression in the horse. Lactate can be taken up from plasma by red blood cells (RBCs). In horses, two isoforms, MCT1 and MCT2, and the ancillary protein CD147 are expressed in RBC membranes. The horse is the only species studied in which RBCs have been found to express MCT2, and the physiological role of this protein in RBCs is unknown. The majority of horses express all three proteins, but 10-20% of horses express little or no MCT1 or CD147. This leads to large interindividual variation in the capacity to transport lactate into RBCs. Here, the expression level of MCT1 and CD147 was bimodally distributed in three studied horse breeds: Finnhorse, Standardbred and Thoroughbred. The level of MCT2 expression was distributed unimodally. The expression level of lactate transporters could not be linked to performance markers in Thoroughbred racehorses. In the future, better performance indexes should be developed to better enable the assessment of whether the level of MCT expression affects athletic performance. In human subjects, several mutations in MCT1 have been shown to cause decreased lactate transport activity in muscle and signs of myopathy. In the horse, two amino acid sequence variations, one of which was novel, were detected in MCT1 (V432I and K457Q). The mutations found in horses were in different areas compared to mutations found in humans. One mutation (M125V) was detected in CD147. The mutations found could not be linked with exercise-induced myopathy. MCT4 cDNA was sequenced for the first time in the horse, but no mutations could be detected in this protein.

Cultural differences and capability transfer in cross-border acquisitions: the mediating roles of capability complementarity, absorptive capacity, and social integration

This paper presents an integrative model of the impact of cultural differences on capability transfer in cross-border acquisitions. We propose that cultural differences affect the post-acquisition capability transfer through their impact on social integration, potential absorptive capacity, and capability complementarity. Two dynamic variables – the use of social integration mechanisms, and the degree of operational integration of the acquired unit – are proposed to moderate the effects of cultural differences on social integration and potential absorptive capacity. The implications for acquisition research and practice are discussed.

Use of wetland buffer areas to reduce nitrogen transport from forested catchments: Retention capacity, emissions of N2O and CH4 and vegetation composition dynamics

The use of buffer areas in forested catchments has been actively researched during the last 15 years; but until now, the research has mainly concentrated on the reduction of sediment and phosphorus loads, instead of nitrogen (N). The aim of this thesis was to examine the use of wetland buffer areas to reduce the nitrogen transport in forested catchments and to investigate the environmental impacts involved in their use. Besides the retention capacity, particular attention was paid to the main factors contributing to the N retention, the potential for increased N2O emissions after large N loading, the effects of peatland restoration for use as buffer areas on CH4 emissions, as well as the vegetation composition dynamics induced by the use of peatlands as buffer areas. To study the capacity of buffer areas to reduce N transport in forested catchments, we first used large artificial loadings of N, and then studied the capacity of buffer areas to reduce ammonium (NH4-N) export originating from ditch network maintenance areas in forested catchments. The potential for increased N2O emissions were studied using the closed chamber technique and a large artificial N loading at five buffer areas. Sampling for CH4 emissions and methane-cycling microbial populations were done on three restored buffer areas and on three buffers constructed on natural peatlands. Vegetation composition dynamics was studied at three buffer areas between 1996 and 2009. Wetland buffer areas were efficient in retaining inorganic N from inflow. The key factors contributing to the retention were the size and the length of the buffer, the hydrological loading and the rate of nutrient loading. Our results show that although the N2O emissions may increase temporarily to very high levels after a large N loading into the buffer area, the buffer areas in forested catchments should be viewed as insignificant sources of N2O. CH4 fluxes were substantially higher from buffers constructed on natural peatlands than from the restored buffer areas, probably because of the slow recovery of methanogens after restoration. The use of peatlands as buffer areas was followed by clear changes in plant species composition and the largest changes occurred in the upstream parts of the buffer areas and the wet lawn-level surfaces, where the contact between the vegetation and the through-flow waters was closer than for the downstream parts and dry hummock sites. The changes in the plant species composition may be an undesired phenomenon especially in the case of the mires representing endangered mire site types, and therefore the construction of new buffer areas should be primarily directed into drained peatland areas.

The roles of nitrification and nitrate reduction pathways in nitrogen cycling of Baltic Sea

The Baltic Sea is one of the largest brackish water bodies in the world. Primary production in the Baltic Sea is limited by nitrogen (N) availability with the exception of river outlets and the northernmost phosphorus limited basin. The excess human induced N load from the drainage basin has caused severe eutrophication of the sea. The excess N loads can be mitigated by microbe mediated natural N removal processes that are found in the oxic-anoxic interfaces in sediments and water column redoxclines. Such interfaces allow the close coupling between the oxic nitrification process, and anoxic denitrification and anaerobic ammonium oxidation (anammox) processes that lead to the formation of molecular nitrogen gas. These processes are governed by various environmental parameters. The effects of these parameters on N processes were investigated in the northern Baltic Sea sediments. During summer months when the sediment organic content is at its highest, nitrification and denitrification reach their maximum rates. However, nitrification had no excess potential, which was probably because of high competition for molecular oxygen (O2) between heterotrophic and nitrification microbes. Subsequently, the limited nitrate (NO3-) availability inhibited denitrification. In fall, winter and spring, nitrification was limited by ammonium availability and denitrification limited by the availability of organic carbon and occasionally by NO3-. Anaerobic ammonium oxidation (anammox) was not an important N removal process in the northern Baltic Sea. Modeling studies suggest that when hypoxia expands in the Baltic Sea, N removal intensifies. However, the results of this study suggest the opposite because bottom water hypoxia (O2< 2 ml l-1) decreased the denitrification rates in sediments. Moreover, N was recycled by the dissimilatory nitrate reduction to ammonium (DNRA) process instead of being removed from the water ecosystem. High N removal potentials were found in the anoxic water column in the deep basins of the Baltic Proper. However, the N removal in the water column appeared to be limited by low substrate availability, because the water at the depths at which the substrate producing nitrification process occurred, rarely mix with the water at the depths at which N removal processes were found. Overall, the natural N removal capacity of the northern Baltic Sea decreased compared to values measured in mid 1990s and early 2000. The reason for this appears to be increasing hypoxia.

Anaerobic ammonium oxidation (anammox) in sediments of the Gulf of Finland

Anaerobic ammonium oxidation (anammox) and denitrification were measured in the open sea and coastal accumulation basins of the Gulf of Finland. The different methods used gave conflicting results on the importance of the anammox process in the sediments. Anammox generally contributed less than 20 % to the total N-2 production, and no anammox was found in a shallow inner estuary basin. However, the discovery of the anammox process in the open sea sediments challenges the denitrification measurements made in the area, as the coexistence of anammox and denitrification compromises the central assumptions behind the method used in denitrification measurements and causes overestimates of the N-2 production. The high (NO3-)-N-15 incubation concentration used in Baltic Sea denitrification measurements exacerbates this overestimation, which is likely to have been substantial.