Maatalous- metsätieteellinen tiedekunta
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(2016)Coniine, a piperidine alkaloid, is known from poison hemlock (Conium maculatum L.), twelve Aloe species and Sarracenia flava L. Its biosynthesis is not well understood, although a possible route starts with a polyketide formed by a polyketide synthase (PKS). This study focused on identification and characterization of PKS-genes involved in coniine formation, induction of callus from plants containing hemlock alkaloids and investigation of the possibility to elicitate the alkaloid pathway in cell culture in order to understand coniine biosynthesis. Plant materials involved in different stages of this study were investigated for their alkaloid content using gas chromatography-mass spectrometry. A novel type of PKS, CPKS5, was identified as the starter candidate for the initiation of coniine biosynthesis by catalysing the synthesis of the carbon backbone from one butyryl-CoA and two malonyl-CoA moieties. When elicitated, poison hemlock cell cultures produced furanocoumarins but no piperidine alkaloids. The hemlock alkaloids are wider distributed than previously has been thought among Sarracenia, and Aloe spp. contain a new alkaloid for the genus. These results together pave the way towards possible utilization of hemlock alkaloids.
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(Helsingin yliopisto, 2016)Energy saving objectives have arisen recently in all sectors of life due to the climate change sce-narios and depletion of fossil energy resources. In boreal and northern temperate climate zone countries one of the most energy intensive operations in arable farming is grain preservation, which in most cases means drying. This work focused on examining the energy use and energy saving possibilities in grain drying, and grain preservation in general, from several aspects. The main focus was in Finnish conditions, but the results can be applied also in other areas, when applicable. In addition to the energy aspect, some economic considerations were conducted, since economic profit is the ultimate decision making factor for farmers. The principal method for grain preservation in Finland is hot-air drying. Energy use and energy savings in drying hence constituted the major part of the research work included herein. The aim of the work was to produce information that could be easily utilized in current farming practices. Therefore all the experiments and measurements were conducted in grain recirculat-ing mixed-flow hot-air dryer, which is the most common grain dryer type in Finland. The energy utilization in dryer was first examined and energy losses identified, and three energy saving methods were chosen for closer consideration: 1) controlling the air flow rate and temperature of the drying air (publications I and II), 2) heat insulation of the dryer device (III) and 3) heat recovery from the dryer exhaust air (IV). The aim of the method 1) was to improve the utilization of the supplied heat energy by increasing the humidity of the dryer exhaust air and thus reducing the energy losses via sensible heat in the exhaust air. Method 2) aimed to eliminate the heat losses from the dryer surfaces by the heat insulation. Method 3) focused on recovering the sensible as well as latent heat from the dryer exhaust air with a passive parallel plate heat exchanger. In addition to the energy saving potential in drying, also the possibilities for enhanced use of alternative moist grain preservation methods and their effects on the energy consump-tion in grain preservation were examined (publication V). While drying is practically the only suit-able preservation method for market quality grain in Finnish conditions, moist grain preservation methods could be applied basically for all home-grown grain used for animal feeding, which rep-resents roughly one third of the total annual grain yield in Finland. The examined moist grain preservation methods were airtight preservation, acid preservation of whole grains and grain crimping (ensiling). The energy saving possibilities with these methods, compared to the current situation, were evaluated by theoretical calculations. The results indicated that considerable energy savings could be achieved by the methods examined in publications I to V. The drying process control method produced energy savings of 5 15%, depending on the grain species, compared to conventional drying method. The energy savings achieved by the heat insulation were 16 21% in the examined dryer. Heat recovery method saved on average 18% energy compared to the conventional system, and energy sav-ings up to 40% were suggested for passive heat recovery by the theoretical model developed in the publication IV. Moist grain preservation methods for preserving home-grown feed grain provided energy savings of 50 90% compared to drying. The combined energy saving potential of the examined methods was 20 43% of the total energy consumption in grain preservation at the present situation, when the realizable potential was considered. It was concluded that significant energy saving possibilities exist in current grain preser-vation practices and their utilization could aid to reduce the energy consumption in the agricul-tural sector and thus to achieve the energy saving objectives set by authorities. Energy savings of 5 11% of the total direct energy use in arable the farming sector could be achieved. However, even the relatively large energy savings would have quite a modest effect on the economy of farming with the current energy prices, which indicates that the direct energy inputs are still relatively cheap, compared to other inputs. While energy prices are expected to rise in the long term, energy saving measures will become more viable for the economy of farms in the future, which is the ultimate incentive for more energy efficient production.
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(Helsingin yliopisto, 2016)Gluten-free foods are defined by their lack of prolamins that trigger coeliac disease. In order for foods derived from Triticeae grains to be considered as gluten-free, the prolamins need to be eliminated. Previously, enzymatic degradation of cereal prolamins has been intensively studied. In the present work, another method is investigated, namely the metal-catalysed oxidation of prolamins. The aim was to modify prolamin peptides and proteins, and to examine the modifications, in order to reduce their immunological activities. The metal-catalysed oxidation was first examined on model peptides, including repetitive sequences of prolamin peptides and a model coeliac-toxic peptide (33-mer), and then tested on C-hordein, a model monomeric barley prolamin, and finally on whole hordein, thus covering the range from peptides to a whole protein. The fragmentation of prolamin peptides and proteins was observed to be a consequence, partly due to proline oxidation. On the other hand, aggregation also occurred, through dityrosine formation, disulfide bridges or carbonyl interactions. The amino acid profile and protein structure affected the oxidation behaviours and resulted in various modifications that were compared after metal-catalysed oxidation treatments. The immunoreactivity of oxidised peptides, C-hordein or whole hordein decreased in R5 antibody-based measurements that are used in gluten-free detection. The present study demonstrated that cereal prolamins can be modified by metal-catalysed oxidation, and their immunoreactivities against R5 antibody were reduced. The information of their oxidative modifications offers a new alternative to be utilised in the elimination of prolamins for gluten-free applications.
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(Helsingin yliopisto, 2016)The wood-decaying white-rot fungi have the profound ability to completely degrade lignocelluloses and all wood components. These fungi and their enzymes have evolved to modify the various lignocellulose feedstocks in nature, and thereby, they are important organisms for bioconversions as well as in fundamental research on fungal biology. The enzymes have many potential applications in biotechnology and industrial purposes including bioenergy production. Evolutionary background of the fungal species and their organelles thus requires deeper understanding to aid in elucidating the relationship of the species to their lifestyles. This PhD study concentrated on the white-rot fungal species Phlebia radiata, Finnish isolate number 79 (FBCC0043). The phylogenetic studies confirmed positioning of P. radiata species in the systematic class Agaricomycetes of Basidiomycota, and in the phlebioid clade of the order Polyporales. The sequenced and annotated mitochondrial genome of P. radiata was discovered to have features that indicate evolutionary pressure and structural diversity in fungal mitogenomes, not being as stable and compact entities than was previously believed. In this study, P. radiata together with species like Phlebia acerina and Phlebia brevispora was demonstrated to form a Phlebia sensu stricto group which consists of efficient producers of lignin-modifying enzymes. The results pinpointed that there is a species-level connection of fungal molecular systematics to the efficiency in the production of wood-decaying enzymes and activities. Norway spruce (Picea abies) is a common tree species in the boreal forests providing an important source of biomass for forest-based industry. Therefore, P. radiata was cultivated on Norway spruce wood under conditions mimicking natural solid-wood colonization, up to six weeks of growth, and the dynamics of fungal enzyme production and gene expression was studied. The lignin-modifying class-II peroxidases (LiPs and various MnPs) were produced, especially in the beginning of fungal growth and colonization of wood, thus indicating the essence of class-II peroxidase as the primary enzymes to function against coniferous wood lignin. Moreover, these extracellular oxidoreductases enhance the accessibility of lignocellulose carbohydrates and thereby, they promote fungal growth in wood. Simultaneously, lytic polysaccharide monooxygenases and several CAZyme glycoside hydrolases attacking cellulose, hemicellulose and pectin were produced, which demonstrates ongoing depolymerization of the polysaccharides to monomers and oligomers. Electron microscopic examination of fungal-colonized wood after six weeks of growth indicated that the decay of wood cell walls was initiated at the tracheid lumen side apparently proceeding towards the middle lamellae. Furthermore, degradation of spruce wood lignin was detected by pyrolysis-GC/MS as decrease in the amount of phenylpropane units with concomitant increase in the number of smaller fragmented products from these lignin units. Thus, the previously observed unique and strong ability of P. radiata to degrade wood lignin and lignin-like aromatic compounds was confirmed. According to the results of this PhD study, P. radiata produces the white-rot type of decay of wood components when growing on Norway spruce. This is due to the efficient ability of the fungus to express and produce a versatile enzyme repertoire for degradation of wood lignocellulose, and in consequence, to generate diverse reactions and bioconversions important for carbon cycling in the forest ecosystems.
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(Helsingin yliopisto, 2016)Cycling of carbon (C) and nutrients plays pivotal role for functioning of every ecosystem. Biogeochemical cycles of carbon and nitrogen (N) are balanced by a network of inter-actions between plants, litter and soil chemistry, microbial communities, enzyme machinery and climate conditions. This thesis focuses on the role of terpenes in C and N transformations in boreal forest soils. Terpenes are abundant plant secondary compounds. The focus was on certain mono-, di-, and triterpenes. Soil incubation experiments revealed that terpenes increased the mineralization of carbon but decreased net nitrogen mineralization and net nitrification. Additionally they increased the amounts of carbon and nitrogen in the microbial biomass through enhancement of bacterial growth; however, they inhibited fungal growth. This study suggests that terpenes can act as a C source for some microbial communities. Moreover, terpenes showed inhibitory potential against enzymes, which are involved in C, N, P, S cycling. The mechanism of inhibition seems to be based at least partially on ability of terpenes to bind enzymes. The field experiment presented the effect of logging residues and wood ash on composition of terpenes and C and N cycling in soil five years after clear-cutting a Norway spruce stand. Logging residue treatment increased the concentrations of certain terpenes in the organic layer. Both, logging residue and wood ash treatments increased net N mineralization and net nitrification. Some changes in terpene concentrations correlated with C and N cycling processes, but the relationship between terpene concentration and C and N cycling processes remained still unclear in the field conditions. In conclusion, terpenes can affect C and N transformations in boreal forest soil. It is probable that terpenes change N cycling retaining more N in organic forms and potentially decrease nitrogen losses from forest ecosystem.
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(Helsingin yliopisto, 2016)Many lactic acid bacteria are able to synthesize dextrans from sucrose by dextransucrases. Weissella confusa strains have attracted increasing attention due to their production of texture-modifying dextrans during food fermentation. Potential prebiotic oligosaccharides have also been produced by dextransucrases in the presence of sucrose and acceptor sugars. However, only few W. confusa dextransucrases have been previously studied, and the reactions of Weissella dextransucrases to synthesize oligosaccharides have not been investigated. In this thesis, two W. confusa dextransucrases from efficient dextran-producers were studied, and their products were structurally characterized because this information is essential for a better usability of these enzymes and corresponding bacteria in food and health applications. The biochemical and kinetic properties of one of the W. confusa dextransucrases were studied. Two activity assays were compared to determine the kinetic parameters. A sucrose radioisotope assay gave a KM of 14.7 mM and a Vmax of 8.2 µmol/(mg∙min), whereas a Nelson-Somogyi assay gave values of 13.0 mM and 19.9 µmol/(mg∙min), respectively. The dextrans from the two W. confusa dextransucrases were found by, e.g., NMR analysis to consist of mainly α-(1→6) linkages and 3% α-(1→3) branches, of which some were elongated. A high-performance size-exclusion chromatography analysis of the dextrans revealed high molar masses of 107‒108 g/mol. Weissella dextransucrases were also studied with acceptor sugars for the synthesis of glucooligosaccharides. The most efficient acceptor was maltose, followed by isomaltose, maltotriose, and nigerose, which formed series of glucooligosaccharides by the further elongation of intermediate acceptor products. The products derived from maltose formed two homologous series, with one series being predominant and the other being minor. The major maltose product series were linear isomaltooligosaccharides (IMOs) with reducing-end maltose units, as identified by multistage mass spectrometry (MSn) analysis. The minor maltose series were revealed by an NMR analysis of the isolated minor trisaccharide product to bear a novel branched structure. These products contained an α-(1→2)-linked glucosyl residue on the reducing residue of the linear IMOs. These structures have not been previously obtained by a dextransucrase. They probably formed by the attachment of a single-unit branch to linear IMOs. For the acceptor analogs lactose and cellobiose, their main acceptor products were identified by NMR analysis to be branched trisaccharides, with a glucosyl residue α-(1→2) linked to the acceptor s reducing end. Surprisingly, a side product, isomelezitose (6Fru-α-Glcp-sucrose), was produced when using lactose as an acceptor. The synthesis of this nonreducing trisaccharide by a dextransucrase was reported here for the first time. Linear IMOs with a degree of polymerization ≥3.3 serve as prebiotics for their resistance to digestion. However, industrial IMO production often leads to a high portion of unwanted digestible sugars. The dextransucrase reaction in the presence of the efficient acceptor maltose was demonstrated here as a promising alternative synthesis process to control IMO size distribution by varying the sucrose/maltose ratio. The effects of substrate concentrations (0.15 1 M) and dextransucrase dosage (1 10 U/g sucrose) on the IMO yield and profile were modeled. High sucrose (1 M) and medium maltose (0.5 M) concentrations were found to be optimal for the synthesis of long-chain IMOs, with 366 g/L of total IMOs attained.
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(Helsingin yliopisto, 2016)Agricultural lands are managed or modified ecosystems that interact with the surrounding natural environment so as to supply while also to use a great range of ecosystem services (ES). In addition, agriculture is responsible for disservices that negatively affect natural ecosystems. In Finland, agricultural lands have undergone marked changes affecting a great number of vital ES. Traditional agri-environmental policy has been criticized for being inefficient in ensuring the provision of ES or limiting the disservices, while markets have been unable to reflect the demand for or supply of ES due to the public character of some ES. Market-based mechanisms as well as targeted policy measures may ensure effective and efficient ES provision. This dissertation explores the factors that determine the citizen demand for and landowner supply of ES, and considers examples of market- and target-based measures that may supplement or replace the current form of agri-environmental policy. The first part of the dissertation is focused on the demand for and supply of cultural ES provided by agricultural lands. A choice experiment was applied to evaluate a marketbased scheme, i.e. a Payment for Ecosystem Services (PES) scheme that provides certain landscape attributes in a typical agricultural area. The analysis revealed that the most valued attributes were the renovation of production buildings and the presence of grazing animals. The results demonstrated that citizen preferences were heterogeneous, a fact which may affect the level of transaction costs and the performance of the scheme. Landowners were skeptical towards the scheme, willing to provide ES that did not always match with the demand. They also demanded compensation in excess of their expenses. Nonetheless, cost benefit considerations revealed that the scheme may be feasible, as the aggregated welfare benefits outweigh the anticipated costs. The second part is focused on the supply of water conservation services and the avoidance of water eutrophication disservices. During the data collection, Finnish agrienvironmental policy set equal incentives for water conservation, not accounting for environmental conditions, which are spatially varied. Before suggesting any policy reform and the use of alternative measures such as target-based measures, where farmers are compensated for delivering certain ES, it is imperative to investigate the tendency of landowners to adopt water conservation measures. By combining survey data with GIS data, a binary choice model was employed. The model examined the adoption of special measures for water conservation if the soil quality implies a high leaching risk and if the water quality is already poor. Adoption in areas under risk was weakly supported by the study s estimates. This indicates that environmental awareness, assuming it increases with risk, is not strong enough to motivate adoption. Target-based which are spatially tailored measures can attract adopters in hotspot areas. The latter outcome leads to the last subject, which examines farmers participation in an agri-environmental auction scheme. According to the outcomes of the study, farmers who have previously participated in a pilot auction scheme were more likely to be participants in future auctions. The findings also suggested a strong relationship between attitudes and participation, particularly for attitudes related to specific environmental benefits attached to the auction scheme, novelty and financial features, as well as the complexity of the auction mechanism. The ES and disservices examined in this dissertation, i.e. landscape amenities and water eutrophication, are of priority. Finnish agricultural landscape has experienced severe stresses during the past years while the state of the Baltic Sea is largely affected by the eutrophication issues of water bodies. The empirical research findings enhance current knowledge in planning market- and target-based schemes in the years to come. These schemes are attracting increasing attention for being more effective and, if properly designed, more efficient. For agri-environmental auctions in particular, the findings are novel, since they were derived from the first auction experiment ever implemented in Finland.
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(Helsingin yliopisto, 2016)Strawberries (Fragaria sp.) belong to the large family of Rosaceae that includes commercially important crop plants such as apple, pear, peach and roses. The economic impact of these species is huge and breeders around the world are striving to keep up with consumers' demands on novelty produce. At the same time, climate change is having an impact on the onset of flowering especially in species that are grown in temperate climates. As flowering is a prerequisite for yield formation, it is extremely important to gain an insight on how the environmental factors, most importantly photoperiod and temperature, affect the timing of flowering in Rosaceous species. Although studying flowering responses directly in the cultivated species could provide immediate practical applications, it is often not feasible due to e.g. complex genomics of the species, large plant size or long juvenile period. The woodland strawberry (Fragaria vesca) (L.) has arisen as a convenient model plant for strawberries and the entire Rose family. It is a diploid species and therefore has a less complex genome than the cultivated octoploid strawberry. The work described here begun by elucidating the molecular identity of SEASONAL FLOWERING LOCUS (SFL), a locus controlling the switch from seasonal to continuous flowering habit in woodland strawberry. SFL was identified as the woodland strawberry orthologue of TERMINAL FLOWER1 (FvTFL1) based on its location on the strawberry genome and similarity to its Arabidopsis counterpart, TFL1. In woodland strawberry, FvTFL1 was shown to be photoperiodically regulated, and it was demonstrated that the continuous flowering habit is caused by a mutation at FvTFL1. In the following experiments, altered regulation of FvTFL1 was associated with the unique vernalisation requirement in the artic F. vesca accession Alta-1, suggesting a previously uncharacterised function for a TFL1 orthologue. The findings on FvTFL1 were extended to cultivated strawberry. It was demonstrated that F. × ananassa homologue of TFL1 (FaTFL1) also represses flowering, and that differences in the regulation of FaTFL1> were associated with different flowering times in strawberry cultivars. The finding that FaTFL1 is a major determinant in the flowering response of cultivated strawberry provides breeders with a new breeding target; producing cultivars with lowered FaTFL1 expression level could expand the flowering and fruiting season of strawberries. These results clearly demonstrate the feasibility of the model plant approach and also highlight the importance of fundamental research. The knowledge gained on fundamental genetic pathways in model plants can be transferred to crop plants, in which similar genetic studies would be impossible or at least extremely complex, slow and costly to perform
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(Finnish Society of Forest Science, 2016)The forest sector has been able to develop many new bio-based and sustainable products catered to business-to-business markets, but the sector still lacks a breakthrough in new forest products and services targeting consumer markets. This is due to higher prices of forest products compared to competing materials, such as concrete, steel, and plastics, but also due to a lack of new product and service innovations targeting end-consumer markets. To understand the emergence of bioeconomy, also bringing new consumer market opportunities to the forest sector, we need to understand the new business ecosystem. The business ecosystem model is a holistic view of the current institutions capturing stakeholder relations and opportunities brought by various resources and technologies. Better understanding of these concepts can lead to consumer-driven development of forest products and services, and improved competitive advantage. This doctoral dissertation introduces a holistic research and development model for new product and service innovations in the forest sector. This research was motivated by the fact that recent forest sector product introductions have been driven by the technology push, and therefore, this research concentrates on the consumer perspective to build new business models and the development of products and services to meet current consumer trends. Evolving sustainability trends among consumers, where bio-based forest products and services can fulfill consumer demand, are central in this thesis. The doctoral thesis consists of one book chapter and three peer-reviewed articles, each using different methodologies. The subjects and results of the papers are grouped into three themes in the framework of the Consumer-Driven Business Ecosystem Research and Development (CDBERD) model. The model extends the classical technology-push and demand-pull innovation model, to better consider consumer values, enabling resources and dominant logics along with the smooth flow of information during each phase of the research and development process leading towards new consumer-driven solutions.
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(Helsingin yliopisto, 2016)The concept of ecosystem services, wide-spread in academia and policy making, emphasizes societal and economic dependence on natural systems for, among others, provision of food, fibres and water, regulation of climate and soil, and contribution to spiritual and cultural values. Anthropogenic pressures driven by rapid economic development are, however, causing a disruption in the benefits that humans obtain from ecosystems. Several economic sectors both depend on and impact ecosystem services. There is therefore maturing expectation for the private sector involvement in environmental governance worldwide, as firms are increasingly motivated to pursue sustainability for financial or strategic opportunities. This dissertation is among the first to address the current research void on the interactions between industries and ecosystem services. The topic is analysed in the context of extensive plantation forestry in the emerging economy of China. The main aim of this work is to investigate if and how the ecosystem services narrative can contribute to further develop corporate sustainability agendas and practices. Based on the findings from this work, ecosystem services research can provide insights and tools to pursue a more comprehensive and holistic acknowledgement of and response to interlinked ecological and social issues in corporate sustainability. For instance it can enable the understanding of company impacts and dependencies on ecosystems, and associated business risks and opportunities; deepen the analysis of company stakeholders perspectives and expectations; contribute to the design of sustainability-oriented practices; and enrich corporate disclosure practices. Further operationalisation of the ecosystem services approach into corporate sustainability would require a more systematic assessment and comparison of the relations between relevant business sectors and ecosystems, the analysis of global-local trade-offs, the internalizations of concepts such as ecological limits and ecological resilience, and the elaboration of suitable corporate and industrial response strategies. Keywords: China; Corporate sustainability; Ecosystem services; Forest sector; Plantations.
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(Helsingin yliopisto, 2016)Basidiomycete white rot fungi are wood-rotting species and their impact to the global carbon cycle is significant. White rot fungi are capable of degrading all the polymeric cell wall components of the plant biomass from polysaccharides, cellulose, hemicelluloses and pectin, to the aromatic heteropolymer lignin. This is due to their ability to produce diverse set of extracellular enzymes that degrade or modify the plant cell wall concomitantly releasing carbon. Research on plant-biomass-degrading fungi has concentrated on isolation and characterization of enzymes especially from the ascomycete fungi for biotechnological applications, such as bioenergy, food processing and waste treatment. More recently genomic studies have opened the reservoir of the plant-biomass-degrading potential of basidiomycete fungi including wood-rotting, litter-decomposing, plant-pathogenic and ectomycorrhizal species. Dichomitus squalens is a white-rot fungus, which colonises softwood and is able to efficiently degrade lignin and cellulose. Previously, intensive studies on white rot fungi have been focused on lignin degradation by oxidative enzymes. The aim of this study was to analyse the potential of the plant-cell-wall-modifying enzymes of D. squalens. Plant biomass degradation by D. squalens was studied at different levels from gene expression to enzyme production. The focus was to dissect the overall degradation of plant biomass polymers, especially cellulose degrading enzymes of D. squalens. The cellulose degradation by D. squalens was studied at the transcript level during growth on spruce wood sticks and in microcrystalline cellulose-containing liquid medium. Selected cellulases and oxidoreductases, which putatively act on cellulose were expressed simultaneously on spruce, the natural substrate of the fungus, and microcrystalline cellulose in time- and substrate-dependent manner. To clarify the adaptation of D. squalens to different plant biomass, the transcriptome and secretome of the fungus were studied in different wood and non-woody substrates. The study confirmed that lignin degradation occurs at the initial stage of growth and D. squalens has retained the diverse enzyme set both for the degradation of wood and non-woody plant biomass. The cellobiohydrolases (CBHs) and cellobiose dehydrogenase of D. squalens were biochemically characterized. In hydrolysis of different plant-derived biomasses, CBHs released reducing sugars alone and in combination with oxidative laccase enzyme. The study shows that D. squalens encodes a complete enzymatic repertoire for plant biomass degradation. In addition, the data emphasise the role of oxidoreductases in the white rot fungal degradation of cellulose and other plant cell wall polymers. Results suggest that white rot fungal plant cell wall converting enzymes are promising candidates in the biotechnological applications using plant biomass.
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(Helsingin yliopisto, 2016)The aim of the thesis was to investigate the interaction between sarcoplasmic proteins and myofibrillar proteins, considering the effects on the loss of water-holding in mild heat induced denaturation such as occurring in pale, soft and exudative (PSE) condition. Porcine longissimus thoracis et lumborum muscles were incubated at temperature 0, 10, 20, 30 or 40 ˚C to 6 h post mortem. Incubation at 40 ˚C reduced the water-holding of meat compared to the lower temperatures (P < 0.001). SDS-PAGE and Western blot analyses indicated that glycogen phosphorylase and creatine kinase precipitated with the myofilaments, which was already accomplished at 6 h post mortem. Substantial meat tenderization was measured after incubation at 40 ˚C, but with less activity of extracted μ- and m- calpains compared to lower temperatures (P < 0.001), which suggests that an early activation of calpains at the highest incubation temperature could have been the reason for the tenderization. Surface hydrophobicity of myofilanments was higher after the pre-rigor incubation at 40 ˚C compared to lower temperatures (P < 0.001). Less myosin subfragment-1 (S1) units were released by chymotryptic cleavage simultaneously with the loss of Ca2+ ATPase activity after incubation at 40 ˚C than at lower incubation temperatures (P < 0.001). The results suggest that the high temperature incubation induce microstructural alterations on the myosin head (S1) region, which may in turn have been related to the loss of water-holding. The roles of the denaturation of sarcoplasmic proteins and myofibrillar proteins were compared. Sarcoplasm not-incubated or incubated at 44 ˚C were mixed with protein-depleted sarcoplasm in different rations and the mixtures were combined with myofibrils and subjected to PSE-like condition pH 5.6/44 ˚C for 1 h . Water-holding was the poorest without the incubated sarcoplasmic proteins. Precipitated sarcoplasmic proteins shrank the myofilamental lattice spacing by 6.3%, compared to protein-free sarcoplasm, during post-rigor incubation at 44 ˚C, shown by X-ray diffraction. These results challenge the current understanding of the role of the denaturation of different proteins in water-holding, and therefore, a new hypothesis is proposed in this thesis: 1) in the intramyofibrillar space, the presence of precipitating sarcoplasmic proteins is associated with the filamental lattice compression that expels water; 2) in the space outside the myofibrils (intermyofibrillar space) within fiber and up to the extracellular space, the coagulated sarcoplasmic proteins form a network that traps water expelled from the intramyofibrillar space.
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(Unigrafia, 2016)ABSTRACT Studies of the taxonomy of bacteria were initiated in the last quarter of the 19th century when bacteria were classified in six genera placed in four tribes based on their morphological appearance. Since then the taxonomy of bacteria has been revolutionized several times. At present, 30 phyla belong to the domain Bacteria , which includes over 9600 species. Unlike many eukaryotes, bacteria lack complex morphological characters and practically phylogenetically informative fossils. It is partly due to these reasons that bacterial taxonomy is complicated. Due to the improvement of methods to obtain sequence level characters plus new methods for their analyses, the taxonomy of bacteria has also been improved. However, there is still no official classification of prokaryotes. Biological nitrogen fixation (BNF) is a process in which bacteria reduce inert nitrogen gas to biologically useful ammonia. The symbiotic interaction between rhizobia and legumes (Fabaceae or Leguminosae) is important both in natural systems and in agriculture. Rhizobia is a general name for a group of bacteria that can enter symbiosis with legumes. Until 1982, all these were classified into one single bacterial genus, Rhizobium. The number of rhizobial genera increased to 17 by the year 2011, from which five genera, Rhizobium, Allorhizobium, Agrobacterium, Ensifer (syn. Sinorhizobium), and Shinella were accommodated in the family Rhizobiaceae. The genus Agrobacterium, a group of mostly pathogenic bacteria, was placed among the beneficial nitrogen-fixing bacteria (rhizobia) in the family Rhizobiaceae. That resulted in several taxonomic issues regarding the family Rhizobiaceae. The main nomenclatural issue regarding the genus Agrobacterium resulted from transferring this genus to the genus Rhizobium. Moreover, the phylogenetic position of the former nitrogen-fixing Rhizobium galegae complex was not clear. This group of bacteria was in previous studies clustered with either Agrobacterium or Rhizobium or placed in a lineage separately from other genera of the family Rhizobiaceae. During the last decade, the number of the rhizobial species increased dramatically, especially in the genus Rhizobium. However, Rhizobium is an inappropriate genus name for some of the species assigned to the genus. To resolve some of the major taxonomic uncertainties of the family Rhizobiaceae, two separate multilocus sequencing analyses (MLSA) were performed. In the first study, an MLSA of 114 rhizobial strains was performed by using six housekeeping genes (atpD, glnA, glnII, recA, rpoB, and thrC). The first MLSA study was focusing on the phylogeny of the taxa belonging to the former Rhizobium galegae complex and the genus Agrobacterium. In the second MLSA, a total of 100 strains representing 81 species of the family Rhizobiaceae were studied using four housekeeping genes namely 16S rRNA, atpD, recA, and rpoB. Based on these results, we proposed delineation of two new genera, Neorhizobium gen. nov. and Pararhizobium gen. nov., and 16 new species combinations, Neorhizobium galegae comb. nov., Neorhizobium huautlense comb. nov., Neorhizobium alkalisoli comb. nov., Agrobacterium nepotum comb. nov., Agrobacterium pusense comb. nov., Agrobacterium skierniewicense comb. nov., Allorhizobium vitis comb. nov., Allorhizobium taibaishanense comb. nov., Allorhizobium paknamense comb. nov., Allorhizobium oryzae comb. nov., Allorhizobium pseudoryzae comb. nov., Allorhizobium borbori comb. nov., Pararhizobium giardinii comb. nov., Pararhizobium capsulatum comb. nov., Pararhizobium herbae comb. nov., and Pararhizobium sphaerophysae comb. nov. (Paper I and II). A total of 159 bacterial strains were isolated from the nodules of the Chinese specimens of the plant genus Glycyrrhiza L. The results of the study showed that 29 true symbiotic strains belong to the genus Mesorhizobium. To estimate the phylogenetic position of the 29 isolates an MLSA was performed for 59 mesorhizobial strains by using three housekeeping genes 16S rRNA, recA, and rpoB. Moreover, the phylogeny of three symbiotic genes (nodA, nodC, and nifH) of these 59 mesorhizobial strains was investigated. The results of MLSA showed that 21 test strains belong to the species M. tianshanense, M. gobiense, M. temperatum, M. muleiense, M. amorphae, M. alhagi, and M. camelthorni, whereas eight test strains might belong to a novel species of Mesorhizobium. The results of the analyses of accessory genes in this study showed that the mesorhizobial strains isolated from the plant genus Glycyrrhiza have probably acquired some genetic material from other rhizobia co-evolving with Glycyrrhiza and other legumes (Paper III).
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(Hansaprint Oy, 2016)Wetland and deadwood loss have had a profound effect on boreal aquatic and terrestrial ecosystems and their biodiversity. Deadwood-dependent species are one of the most endangered organism groups in the world, while amphibians on the other hand excellently represent the ecological state of wetlands. The boreal region contains a large proportion of the world s wetlands, which have undergone two major alterations during the last 500 years: first the extirpation of beavers and secondly draining during the 20th century. Beavers are well-known ecosystem engineers of the Northern Hemisphere. They modify their surroundings by damming water systems. Damming raises flood waters into the surrounding riparian forest and changes environmental conditions both on land and in water. Ecosystem processes are altered when beavers turn a lotic water system into a lentic one, but the alteration is also evident when beavers modify initially lentic water systems. Organic matter and nutrients are transferred into a wetland from beaver-felled trees and vegetation killed by flooding. The amount of dissolved organic carbon increases during the first three impoundment years, which enhances the growth of aquatic vegetation and the abundance of phyto- and zooplankton, thereby also increasing invertebrate abundances. Luxuriant vegetation and ample plankton and invertebrate populations facilitate frogs, which become abundant in beaver wetlands. The moor frog in particular favours beaver-created wetlands. Flooding and beavers kill trees, producing high amounts of deadwood. The riparian forests of beaver wetlands include much higher deadwood levels than wetlands without beavers. Increased deadwood creates substrate resources for deadwood-dependent species. Snags are a typical deadwood type in beaver wetlands. Calicioids are deadwood-dependent species particularly specialised in inhabiting standing deadwood. The comeback of beavers has aided the restoration of wetlands and deadwood. Beaver wetlands can be seen as carbon and biodiversity hot spots that increase the heterogeneity and hydraulic connectivity of the boreal landscape.
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(Helsingin yliopisto, 2016)Antibiotic resistance has become a serious threat to the efficacy of antibiotics used in human and veterinary medicine. Understanding the abundance and prevalence of antibiotic resistance genes (ARGs) in the environmental resistome is important for maintaining the efficacy of antibiotics and predicting a risk of the ARGs spreading in the environment and moving into previously non-resistant bacteria, including human pathogens. Fish farms are an environmental reservoir of ARGs due to the treatment of fish with antibiotics that also are important for human medicine. The two main topics of this thesis are (1) determining the abundance and diversity of ARGs and mobile elements in sediments impacted by fish farming and (2) investigating the major source of ARGs in the farm sediments in the Northern Baltic Sea. In addition, correlations between ARGs and mobile elements were examined to estimate the potential risk of ARG mobilization in the environment. This study employed a high-throughput qPCR array, which permits quantifying hundreds of ARGs and genes associated with mobile elements in the environmental resistome in a single experiment. Fish farming impacts the composition of ARGs in sediments below fish farms in the Northern Baltic Sea. However, the impact is local and mostly limited to enrichment of ARGs associated with antibiotics used at the farms. In the current conditions, the risk of ARG spread from the farm sediments to the surrounding sediments is low in the Northern Baltic Sea. However, the enriched ARGs persist in the farm sediments during the 6-year observations even when the selection pressure of the antibiotics is negligible. Moreover, significant correlations between mobile elements and ARGs may imply the persistence of certain ARGs in the fish farming environments and their potential for mobilizing the ARGs to other bacteria including pathogens. The persistence of ARGs at the farm facilities is a threat to the efficacy of the antibiotics against fish diseases, potentially leading to fish production losses. We provide indirect evidence suggesting that certain ARGs are being constantly introduced by feces of the farmed fish into the sediments below the fish farms. Further studies could focus on investigating the development of ARGs in juvenile fish before they are introduced into the Baltic Sea open-cage farms. We conclude that a high throughput qPCR array is a powerful tool that provides unprecedented insights into the ARG composition in the environmental resistome associated with fish farming.
