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  • Damerau, Annelie (Helsingin yliopisto, 2015)
    The consumption of whole grain foods high in fibre is of interest because of the health-promoting effects associated with dietary fibre. Therefore, there is interest in developing new fibre-rich cereal foods. However, these kinds of foods also contain polyunsaturated lipids, which are prone to oxidation. Further, lipids are dispersed in a heterogeneous matrix of starch, proteins and fibre, which increases their tendency to oxidize because of a large surface area and possible contact with prooxidants. The oxidation of lipids decreases nutritional quality and causes the formation of undesirable flavours. Knowledge of the oxidation behaviour of dispersed lipids in solid cereal foods, and of how factors like process parameters, structural features of the products and storage conditions affect lipid oxidation, is limited. In this thesis, the oxidative behaviour of foods with dispersed lipids was studied using two model systems. The first model system was a spray-dried emulsion containing sunflower oil encapsulated in a Na-caseinate-maltodextrin matrix, with either non-cross-linked or cross-linked proteins. The stability of the total and surface lipid fractions was determined during storage under different relative humidities (RHs). Further, the effect of RH on the amount of volatiles released from oxidized spray-dried emulsions was studied. The second model system consisted of extruded cereals produced from either whole grain oats or rye bran (coarse or fine) using different extrusion parameters. Their oxidative stability was studied during storage at 40 ºC, after milling and standardization to RH 33%. The primary oxidation was measured by peroxide values in the spray-dried emulsions and by losses of tocopherols and tocotrienols in the spray-dried emulsions and rye bran extrudates. Secondary oxidation was determined based on volatile secondary lipid oxidation products analysed by static head space (SHS-GC-FID) in the spray-dried emulsions and by head space solid-phase micro extraction (HS-SPME-GC-MS) in the extruded cereals. In addition to the oxidation parameters, enzymatic hydrolysis of lipids in the oat extrudates and the fatty acid composition of all models were studied by measuring the neutral lipid and fatty acid profiles, respectively. Increasing the RH improved the oxidative stability of both the total and surface lipid fractions of the stored spray-dried emulsions. This behaviour was mainly linked to the loss of individual powder particles upon caking and collapsing of the matrix at RH 75%. In addition, excess protein may have delayed oxidation via its radical scavenging activity. At RH 54%, cross-linking of the protein slightly improved the oxidative stability. The profiles of the volatile oxidation products from the spray-dried emulsions analysed by HS-SPME were also influenced by the RH. The effect was related to water-induced changes in hydrophilicity, structure and binding ability of the matrix, and to partitioning and solubility of the volatiles. The highest overall amount of volatiles released was obtained at water contents of 3.1% and 5.2% (RH 11% and 33%). The enzymatic hydrolysis of lipids in oats was effectively prevented by extrusion, even at the lowest temperature of 70 °C. The extrusion temperature could be increased to 110 °C without subjecting the lipids to non-enzymatic oxidation. However, by increasing the temperature to 130 °C, lipid oxidation was promoted, which also yielded losses of neutral lipids over time. In the case of the rye bran, the low water content (13% or 16%) in the extrusion of coarse or fine bran led to the most stable lipids during storage. The improved oxidative stability at low water contents in extrusion was connected with the higher formation of Maillard reaction products, which could have acted as antioxidants. The grinding of rye bran prior to extrusion caused a loss of tocols and increased the amounts of Maillard reaction products formed. The oxidative stability of the dispersed lipids was shown to be highly related to water induced physical changes in the matrix structure, which makes controlling the RH in the surrounding atmosphere an important factor in storage. Further, the RH affected the amount of volatile lipid oxidation products released, and this needs to be considered in determining lipid oxidation by HS-SPME. Extrusion was shown to inactivate lipases in oats. For the lipid stability in cereal extrudates, low temperature and low water content during extrusion were shown to be beneficial.
  • Holopainen, Sari (Helsingin yliopisto, 2015)
    The boreal biome harbours a large share of the world s wetlands, and is the main breeding area for several duck species. Breeding habitats strongly influence duck reproduction. In this study, the habitat use of ducks and their breeding success was estimated in a boreal landscape in southern Finland. A review of duck habitat use in boreal wetlands was additionally made. Boreal forest lakes are normally considered stable environments, but from a duck s point of view both resource abundance and habitat quality in the lakes may differ from one year to another. In this study the vegetation of boreal freshwater lakes was found to slowly change during the 20-year study period. Beavers (Castor canadensis) caused pronounced alterations in the lakes by flooding them. Flooding rendered lake vegetation structure more luxuriant and increased the lake use by ducks. In general, duck brood lake use was positively associated with both the coverage of wide belts of tall emergent plants and the abundance of emerging insects and aquatic invertebrates. The relative importance of these factors varied between species. The brood production of common goldeneyes (Bucephala clangula) was regulated by density dependence per se, but fluctuated with food availability per capita. No spatial density-dependent effects were found for the common teal (Anas crecca), but the breeding success was instead explained well by habitat quality variation. Both food and flood abundance had a positive effect, but different variables seemed to operate during different phases of the breeding season. Many aspects of boreal breeding ducks are still poorly studied. Especially lacking are studies concerning duckling survival and the effects of anthropogenic actions on duck habitat use. This research underlines the importance of high quality habitats for breeding duck populations in the boreal landscape. The varying habitat requirements of duck species should be considered when implementing wetland management.
  • Majasalmi, Titta (Helsingin yliopisto, 2015)
    The aim of this dissertation is to assess the accuracy of different ground reference methods used to validate satellite based leaf area index (LAI) and the fraction of absorbed photosynthetically active radiation (fPAR) products. LAI and fPAR are strongly linked, although they principally and practically measure different properties: LAI quantifies the areal interphase between soil and atmosphere, whereas fPAR quantifies the energy available for photosynthesis. Until now, the development of remote sensing based methods to estimate LAI and fPAR in a boreal forest has been hindered by the scarcity of ground data, which is required to validate and develop existing algorithms. The aim of the first part of this dissertation was to assess the impacts of different methodological approaches to estimate LAI in boreal forests, and to validate satellite based LAI products. Results showed that the accuracy of ground based LAI estimates is sensitive to both the retrieval methods and sampling scheme used to collect the optical LAI data. The satellite based measurements of LAI demonstrated a large temporal variability in LAI. The second part of the dissertation focused on measuring and modeling fPAR in a boreal forest. A new scheme for measuring and modeling ground reference fPAR based on photon recollision probability was presented in this dissertation. Ground reference fPAR is usually estimated only for the forest canopy layer. This study is among the first ones to validate the new global satellite based fPAR product called GEOV1 using data of both the forest canopy and understory layers from boreal forests. Results showed that satellite based fPAR products may correspond better with the total fPAR, instead of only the forest canopy fPAR as has often been presumed.
  • Hakojärvi, Mikko (Helsingin yliopisto, 2015)
    Precision farming is a set of advanced technologies and cultivation practices that aim to enhance crop growing conditions in a site-specific manner. Current machinery offers many technical tools for such actions, but information about when and where it is necessary to use the developed machinery effectively is lacking. The use of crop models has been proposed to combine the effects of growing season conditions and field spatial properties. However, in a timely and spatially variable environment, which a field often is, much information about site-specific growing conditions should be available in order for an advanced crop model to reproduce the site-specific growth in a detailed manner. Unfortunately the information from fields has often been very limited, and insufficient for such purposes. Furthermore the set of precision farming tools and the number of growth factors that can be managed is limited. For describing maximal biomass accumulation, a simple crop model was introduced and evaluated in this thesis. The model is mechanistic, and it uses a minimal number of parameters that all are based on physics, chemistry or physiology. The model can be used for calculating the radiation or radiation and water limited biomass accumulation of a C3-crop. A field experiment equipped with continuous measurements was used for model establishment and after model establishment the model was evaluated with a field experiment with various radiation, nitrogen fertilization and precipitation conditions. In both the studies the crop model was found to produce the maximal biomass accumulation when parameter values measured in the experiment were used. The model was applied in a study evaluating the effects of selected site- and depth-specific soil properties on yield variation on three different clay soil fields located in southern Finland. In order to evaluate the effects of selected soil properties under various weather conditions a Monte Carlo method was used with the biomass accumulation model and generated precipitations. The yield variation was evaluated according to temporal mean biomass yield and temporal standard deviation of the biomass yield. For studying the use of the model in a spatial environment, the introduced biomass accumulation model was applied in a simulator built for simulating a fully automated crop farm. In addition, the use of continuous soil moisture measurements for measuring the crop water use and further for biomass accumulation was tested. According to the results, the crop model was capable of simulating the highest biomass accumulation of the crops used in the experiments. This was the case for all radiation-limited simulations and for most of the water-limited simulations. In a few cases the values of the observed soil properties were found to cause too low biomass accumulation in simulations but in such cases the problem was also present in the comparisons of observed soil properties and observed soil water content during the growing season. For future research with the model, the next phase will be to test the model use in precision farming-related decision making. The structure of the model enables its use with other C3-crops than small grain cereals. Therefore testing of the model with other C3-crops could be performed in future research.
  • Itkonen, Suvi (Helsingin yliopisto, 2015)
    Phosphorus (P) intake in Western countries exceeds the nutritional recommendations 2- to 3-fold, and the increased use of food additive phosphates (FAPs) in the food industry has augmented total P (TP) intake; an estimated 10-50% of TP intake comes from additives. However, in food composition databases, the amounts of FAPs may have not been taken into account. Difficulties in measuring true P intake occur also because bioavailability of P differs between foodstuffs; inorganic P from additives is absorbed better in the intestine than natural, organic P. The harmfulness of high P intake to kidney patients has been known for years, and dietary P restriction is used in the treatment of the disease to avoid vascular calcification, which occurs due to calcium-phosphate deposition in the vascular vessel wall. However, recently, concerns have been raised about the role of elevated serum phosphate concentrations in cardiovascular health in the general population, but data on dietary P is scarce. This thesis aims to offer new insights into the potential health risks related to high dietary P intake in Finland by providing knowledge on the bioavailability of P in foodstuffs, and on the association of high dietary P intake with cardiovascular disease risk factors in the general population. In Study I, a new method was developed for analysis of in vitro digestible P (DP), indicating bioavailable and absorbable P. DP and TP contents of certain differently processed cereals were analyzed. The calculated uncertainty of the new analysis method had little effect on the DP contents in the samples. Soured cereals contained more DP than unsoured cereals, and the long processing time increased the amounts of DP. In Study II, the DP contents of selected plant-based foodstuffs with or without FAPs were determined by the analysis method developed in Study I. The analyzed plant-based products contained varying amounts of P, but most P was not digestible, except in FAP-containing foodstuffs (cola drinks and baking powder-leavened muffins). In Study III, the associations between TP intake, P density of the diet (TP intake related to energy intake), FAP intake, and a risk factor of cardiovascular disease, carotid intima-media thickness were investigated in a middle-aged Finnish population (n=546). Linear associations among P density of the diet, FAP intake, and carotid intima-media thickness were found when all subjects were included in the analysis, and between FAP intake and carotid intima-media thickness in females (n=370). However, among males (n=176) no significant associations were found. In conclusion, results of this thesis reinforce the previous understanding about the importance of the source of P when assessing P intake in a population. The plant-based foodstuffs generally contain low amounts of DP, but the FAP-containing plant-based products have relatively higher DP contents than FAP-free products. The method developed for the analysis of DP is reliable but requires validation against an in vivo method before these results can be generalized. Albeit high dietary P intake (P density of the diet and FAP intake) was associated with a cardiovascular disease risk factor, carotid intima-media thickness, due to the cross-sectional design of the study, no causal relationships can be inferred. Thus, the potential adverse effects of highly absorbable FAPs should be further investigated both in intervention and in follow-up studies before final conclusions about the harmfulness of high P intake for the general population can be drawn. To find this out, data on use of FAPs in the food industry and updated information on P contents in foodstuffs are also needed.
  • Nousiainen, Aura (Helsingin yliopisto, 2015)
    The use of pesticides has allowed the efficient use of agricultural soil and provided humans with greater yields and agri-food security. Unfortunately, many pesticides have also adverse effects to the environment or human health, and may end up where they were not intended: the precious groundwater reserves. The use of atrazine, a herbicide used for controlling broad-leaf weeds, was banned in the EU for this reason in 2004, but is still globally one of the most widely used herbicides today. Although atrazine can be completely mineralized by microbes, in the subsurface, slow or incomplete degradation of atrazine is often observed. The ability of atrazine degradation by microbes can be utilized in bioremediation, a technique in which contaminants are removed by microbial activity. This study was undertaken to elucidate the potential use of genetic tools, such as quantitative PCR (qPCR), radiorespirometry, microautoradiography (MAR), clone libraries and genetic fingerprinting methods, in atrazine contaminated soils, and to apply them in atrazine bioremediation. Collaboration with our Indian partner permitted comparison between atrazine treated, cropped agricultural soils and boreal subsoil contaminated two decades ago with residual atrazine from weed control in municipal areas. Four different bioremediation methods, natural attenuation, bioaugmentation, biostimulation, and their combination, were used to reduce atrazine concentration in soil. Atrazine degradation copy numbers often reflected the atrazine degradation potential, indicating their robustness as monitoring tools in different soils. The most efficient bioremediation treatment was bioaugmentation by atrazine degrading bacterial strains Pseudomonas citronellolis or Arthrobacter aurescens, or by an atrazine degrading bacterial consortium: in the agricultural soil, up to 90% of atrazine was degraded in less than a week, whereas in the boreal subsoil, 76% of atrazine was mineralized. In the clone library constructed from boreal soil, several clones related to taxa which include known atrazine degraders were found. In this soil, biostimulation with additional carbon was an efficient treatment at reduced temperature. In general, the efficiency of atrazine removal in different treatments was bioaugmentation and biostimulation > bioaugmentation > biostimulation > natural attenuation. Previous exposure to atrazine was the most influential factor in atrazine disappearance from soil, as recent exposure always correlated with faster atrazine degradation, and greatly affected the composition of the microbial community, elucidated by LH-PCR. These results serve as an example on how soil origin, exposure history, organic content and use must be taken into account while choosing the best bioremediation method. Knowledge on the presence of genetic degradation potential can be helpful in choosing the treatment method. While bioaugmentation removed 90% of atrazine from soil, its application in field scale may be challenging. Our results show, that biostimulation alone may serve as the treatment method of choice, even in the challenging subsoil surroundings where atrazine concentrations are low.
  • Karttunen, Kalle (Helsingin yliopisto, 2015)
    The aim of this work was to study how process innovation can be applied in forest biomass supply chains for reducing costs to add value compared to traditional supply chains. The work consisted of four articles using alternative data and a variety of methods. The process innovation of forest biomass supply chains contains several possibilities. There is a need to identify which processes should be renewed incrementally or completely. The main innovation types determined by the case articles were divided into incremental, radical and network innovation. Achieving cost reduction was possible by innovating traditional forest biomass supply chain processes in a novel way in all cases. The case of network innovation however, presenting the co-operation of an entire supply chain with stakeholders by linking forest management and logistics business systems together in process innovation, provided the highest cost reduction, which can be seen as added value. This is because network innovation includes several structural holes with close connections between processes and systems that offer the possibility of finding more cost reduction potential for the entire supply chain. The main conclusion of this work is that it is not worth implementing innovation solely inside a company´s own activities, but opening the innovation process for the whole network of a supply chain is crucial. The methods presented in this work could be mainly applied in forest biomass supply chain innovation. The work enhanced the knowledge of innovation usage for forest biomass supply chains.
  • Chong, Sun-Li (Helsingin yliopisto, 2014)
    O-acetylglucuronoxylans (AcGX) are the major hemicelluloses found in the secondary cell wall of dicotyledon species. The backbone is formed by β(1→4)-linked xylopyranosyl (Xylp) residues, which are substituted by α(1→2)-linked (4-O-methyl)glucopyranosyluronic acid ((Me)GlcpA). The AcGX are also highly acetylated on the 2-O or 3-O; or both positions of Xylp units. Notably, acetylation patterns in AcGX are not well understood since they are typically destroyed during the alkaline isolation. Accurate quantitation of MeGlcpA is also challenged by the lack of commercially available MeGlcpA sources, thus the accuracy of MeGlcpA content determined with the GlcpA standard is unknown. The current thesis established new procedures of detailed characterization of AcGX. The xylan OLIgosaccharide Mass Profiling (OLIMP) method encompassed endoxylanase hydrolysis and mass spectrometry detection. The endoxylanase cleaves the xylan backbone into acetylated xylooligosaccharides (AcXOS). As the action is hindered by the side groups, endoxylanase can be a selective tool to liberate AcXOS from plant tissues for fingerprinting the acetylation pattern in AcGX. Additionally, mass fragmentation analyses were performed to elucidate the spatial distribution of acetyl residues. The accuracy of MeGlcpA quantitation using the GlcpA standard was examined by comparing it to the in-house purified MeGlcpA in acid methanolysis and gas chromatography (GC) analysis. Several of the genes responsible for the biosynthesis of AcGX were previously identified using Arabidopsis thaliana as the model plant. Herein, the structures of AcGX in Arabidopsis wild-type and biosynthetic mutant plants that are defective in reducing end tetrasaccharide sequence or backbone synthesis, irx7, irx9, irx10 and irx14; and (Me)GlcpA addition, gux1gux2 were analyzed using methods established for structural characterization of AcGX. Mono-acetylations (2-O or 3-O position) were reduced in irx7, irx9 and irx14, whereas 2-O acetylation was elevated in the (Me)GlcpA deficient mutant, gux1gux2, indicating that the addition of (Me)GlcpA residues is taking place before acetylation of xylans. Structural elucidation on the major AcXOS liberated from wild-type plant suggests that the acetyl residues are added in every other Xylp residue in AcGX. Interestingly, a novel pentose substitution on the GlcpA side group in AcGX was identified. In acid methanolysis, the GlcpA standard was partially lactonized, thus yielding six derivatives in GC chromatogram. When all six GlcpA derivatives were used in the calibration curve, the MeGlcpA content was underestimated by nearly 30%. The MeGlcpA content can be closely estimated by choosing the appropriate GlcpA derivatives in the calibration curve. The method was used to investigate the impact of Schizophyllum commune glycoside hydrolase family 115 α-glucuronidase (AGU) transgene expressed in Arabidopsis. The (Me)GlcpA content in the ScAGU115 expressing plants was surprisingly unchanged despite the active recombinant enzyme present within the cell walls. In this work, the acetylation pattern in the AcGX of Arabidopsis wild-type and mutant plants was studied in detail. The methods developed herein revealed that the acetylation of AcGX was reduced in Arabidopsis lines that encode a defective biosynthetic gene related to backbone or reducing end sequence synthesis, suggesting pleiotropic effect of a single gene mutation in xylan biosynthesis. The MeGlcA substituents in AcGX can be effectively reduced by disruption of the glucoronyltrasnferases; however, the substitution in the AcGX of gux1gux2 was compensated by acetylation. On the other hand, constitutive transgenic expression of ScAGU115 α-glucuronidase did not remove the (Me)GlcpA substituents in planta. The reason may be ascribed to the shielding by neighbouring acetyl substituents, or limited accessibility of the recombinant enzyme to cell wall substrates. Therefore, a viable approach for the effective tailoring of AcGX substituents in planta could be co-expression with an acetyl xylan esterase to obtain synergism between these side-group removing enzymes.
  • Leppälammi-Kujansuu, Jaana (Finnish Society of Forest Science Finnish Forest Research Institute Faculty of Agriculture and Forestry at the University of Helsinki School of Forest Sciences at the University of Eastern Finland, 2014)
    Knowledge of the quantity of belowground litter carbon (C) input is scarce but highly valued in C budget calculations. Specifically, the turnover rate of fine roots is considered to be one of the most important parameters in the estimation of changes in soil C stock. In this thesis Norway spruce (Picea abies L. (Karst.)) fine root lifespan and litter production and their responses to nutrient availability and temperature were examined. Aboveground foliage and understory litter C inputs were also quantified. Furthermore, fine root isotopic C ages were compared to fine root lifespans. Increased nutrient availability and higher temperature shortened spruce fine root lifespan both in the long-term manipulation treatments and along a natural latitudinal gradient. Fertilization improved tree growth and the absolute amount of litter production, both below- and aboveground. Soil warming, by contrast, increased the belowground litter production in relation to aboveground foliage litterfall but did not lead to long-term increases in aboveground tree growth. In warmed soil, the changes in spruce fine root tip morphology pointed to nutrient deficiency. The results indicated that in nutrient limited forests climate warming is unlikely to increase the aboveground tree growth in the long-term. Fine root litter C input into the soil in relation to the aboveground litter C input was higher towards lower fertility, due particularly to the greater contribution of understory vegetation. The structural 14C age of fine roots was consistently 3 - 6 years older than the fine root lifespan determined with the minirhizotron method, indicating that root growth may use also use stored or recycled C. In almost all stands, fine root litter C input into the soil at least equalled the aboveground input, which confirms the significance of belowground litter production in the boreal forest C cycle. The importance of understory vegetation was also significant. In addition on understory vegetation, different stand age and tree species, more studies should also focus on the shift in the litter production pattern from above- to belowground along environmental change as this may have an impact on litter C quality and soil C storage in boreal forest soils.
  • Alitalo, Anni (Maa- ja elintarviketalouden tutkimuslaitos MTT, 2014)
    Manure serves as an important source of nutrients, but is also an environmental concern. From the sustainable agriculture point of view it is of great importance to develop manure treatment technologies to recycle manure nutrients, and to reduce the odor and hygienic problems due to manure production and use. However, the development work has been greatly hampered by the complexity of manure as a raw material and requires knowledge of various physicochemical and biological factors involved in its utilization. In this study, a sequential slurry manure processing scheme is described in which biological treatment was carried out in a series of continuously fed low-aerated tank reactors. Biological treatment was followed by ammonia stripping, conducted as a sequential stripping procedure. The biological treatment served as a means of achieving a pH increase of manure and made it possible to separate part of the nitrogen by stripping without chemical use. Limited aeration during the biological treatment was applied in order to maintain nitrogen in ammonia form, thus preventing nitrate formation and excessive CO2 formation, and instead favoring humification pocesses. Efficient odor reduction and good hygienic status were also among the targeted objectives of the biological treatment. The functionality of the biological treatment was examined by using swine and dairy slurry manure. Experiments were carried out in pilot scale using six 600-L tanks connected in series with feedback. Before the actual start of the treatment, the treatment tanks were filled with microbial seeding material. It was shown that the designed reactor system provided stability for the process and increased treatment efficiency. However, efficient pretreatment was required in order to reduce the dry matter content of the slurry manure to a level of 1−2% before the biological treatment. Part of the manure phosphorus was also removed with pretreatment. The introduced treatment system reduced the manure odors to an undetectable level or only very faint odor in four days. The pH-value at the end of the treatment increased above 8.5, and nitrogen changes occurred mainly due to ammonia volatilization during the treatments. Nitrate formation was very low with the aeration rates used. Carbon reduction during the aeration treatment depended on the initial carbon content of the manure and varied in the different studies between 11% and 57%. The treatment also caused changes in slurry precipitation characteristics, which were observed as a color change and a decline in the concentration of divalent cations and total phosphorus. It was also shown that the six tanks in series configuration with feedback served as a good device to achieve good hygiene of the end product. At best, over 90% reductions were observed in the numbers of enteric indicator organisms. However, the result obtained varied depending on the treatment run and indicator organism. It was shown that the buffer system in manure slurry was composed of TAN (total ammoniacal nitrogen), CO2, HCO3− and CO32− and thus can be circumvented by making use of the ammonium-carbonate reduction obtained by biological treatment. Over 30% TAN removal by air stripping was shown to be possible without use of chemicals, if the pH of the biologically treated swine manure was above 8.9. It can be concluded that this study explains mechanisms and provides a basis for technologies used to efficiently reduce the manure odor, improve manure hygiene, and to separate nitrogen and phosphorus, enhancing the availability of manure fractions as fertilizer. The presented treatment method provides an option to increase recycling of the nutrients of manure and to reduce the odor and hygienic problems associated with manure slurry.
  • Haapanen, Reija (Helsingin yliopisto, 2014)
    This dissertation explored the potential of image features derived from remotely sensed data in the context of large-area forest inventory. The study areas were located in Finnish boreal forests, with one exception in Northern Minnesota, USA. Estimation of forest variables was carried out at pixel (or an equidistant grid) level. The non-parametric k nearest neighbour estimation method was applied throughout the study. The used remotely sensed data included Landsat 7 Enhanced Thematic Mapper Plus (ETM+) satellite images, colour infra-red aerial photographs, TerraSAR-X radar and airborne laser scanning (ALS) data. An indicative suitability order of these image types for estimation of forest variables was ALS, TerraSAR-X, aerial photographs and Landsat 7 ETM+. Special emphasis was placed on combining features extracted from individual remotely sensed data sources and searching for sets of image features that led to the best performance for estimation of forest variables. Selection of the image features was mainly carried out using a genetic algorithm. The resulting relative root mean square errors (RMSEs) ranged from 23% to 77% in the case of estimating mean volume of growing stock. The best results were obtained employing ALS and aerial photograph-based feature combinations. These combinations led to relative RMSEs of 23 30% when estimating mean volume of growing stock, depending on the landscape complexity. Combining image types with complementary properties typically improved the estimation accuracy. Automatic selection of image feature sets greatly reduced noise and dimensionality of the large feature sets used as input data and resulted in better performance in terms of estimation error. In studies employing ALS data, the ALS observations describing the vertical structure of forest stands played a critical role in decreasing the estimation error.
  • Matilainen, Kaarina (2014)
    Inference for the variance components in linear mixed effects models is theoretically well understood. Many methods have also been presented for nonlinear models. Genetic evaluations in animal breeding are however characterized by the enormous size of the models and data. This means that the methods in estimation have to be computationally efficient. The purpose of this study was to find efficient methods for the estimation of the variance components for large data sets and complex mixed effects models in animal breeding. The focus of the study was, first, on the restricted maximum likelihood (REML) estimation applied to a linearized model of nonlinear mixed effects model and, second, on the REML estimation of large linear mixed effects models by the Monte Carlo (MC) method. Performance of the methods were mostly studied using simulated data sets, but the feasibility of the MC based expectation maximization (EM) REML was also studied using dairy cattle field data. The analyses of a data set mimicking pig live weights showed that linearization works moderately well when the data is good, but estimation of parameters related to adult weight becomes unstable when weight observations from the right tail of the animals growth curve were missing. However, the simulation study showed that having even a small proportion of animals with adult weights improved the results when compared to the estimates based on observations from prematurely slaughtered animals only. The MC based EM REML method converged to the same solutions as the analytical EM REML, and a small number of MC samples did not introduce systematic bias to the estimates of genetic parameters in the analysis of simulated dairy cattle data set. Furthermore, analyses of field data proved the MC EM REML to be superior to the analytical EM REML both in computing time and in the memory needed. Compared to MC EM REML, the MC Newton-type methods converged faster, but sampling variation of the estimates increased. Sampling variation differed somewhat also between the Newton-type methods. Developing a fast algorithm for MC based REML estimation requires a convergence criterion that is robust for sampling variation. A stopping rule that can be calculated during the analysis was introduced. The applied convergence criterion monitored the progress of convergence and was only a little influenced by MC noise. It also worked reasonably well with small number of MC samples, which is a property that may be useful for analyzing large scale and complex models.
  • Liedert, Christina (Helsingin yliopisto, 2014)
    The known natural habitats of Deinococcus geothermalis are geothermal springs and deep ocean subsurfaces. The bacterium has also found its way to manmade environments, including paper machines and drinking water distribution systems, from which it is very difficult to remove due to its resistance towards industrial washing procedures, dehydration and even high doses of ionizing radiation. D. geothermalis attaches tightly on industrial, even microbially repellent, surfaces initiating slime accumulation. We show that the adhesion of D. geothermalis is mediated by outercellular appendages identified as glycosylated type IV pili. D. geothermalis is the first taxon member of the ancient phylum Deinococcus-Thermus, where glycosylated pili are reported. We propose that D. geothermalis is an obligate microaerobic bacterium. Proteomic analysis showed that D. geothermalis, when cultivated aerobically in nutrient limiting medium prevailing in nature, suffered from oxidative stress. Cells did not grow or respire, unless divalent manganese was supplied. As a novel finding, we reported that the cells secreted succinic acid to the medium, possibly as an end product of metabolic pathways directed to battle oxidative stress. The data suggests that D. geothermalis can grow in aerobic habitats, such as the splash areas of paper machines, when divalent manganese is available. Generation of biofilms may be a physiological response of D. geothermalis to escape atmospheric oxygen. Under aerobic conditions D. geothermalis was intolerant to sodium ions beyond 90 mM. Sodium may have inhibitory effect on V-type ATPases, which D. geothermalis uses as its sole mechanism of oxidative phosphorylation. The findings in this thesis offer novel antifouling strategies in processes where D. geothermalis is harmful: (1) destroying the pili by antifouling treatments; (2) promoting dispersal of D. geothermalis from the biofilm by environment-compatible procedures, such as providing process waters with manganese (0.5 mg L-1) or sodium ions (0.5 % w/v), or (3) by reducing dissolved oxygen to levels where D. geothermalis chooses to grow in suspended form rather than biofilms. Alternatively, the description of the D. geothermalis proteome, physiology and biofilm formation may be applied in the development of biotechnological applications where this robust, non-pathogenic and easy-to-culture bacterium is utilized for demanding jobs such as bioremediation of nuclear waste lands or as a resistant host to produce industrially relevant substances.
  • Deegan, Kevin Cárthaigh (Helsingin yliopisto, 2014)
    Homogenisation is a physical process which results in the reduction in the size of fat-globules to reduce creaming. It is not generally used in cheesemaking as it allows access of the indigenous enzyme lipoprotein lipase (LPL) to the triacylglycerol core of the fat globule, resulting in uncontrollable lipolysis and rancidity both in the milk and cheese made therefrom. It was hypothesised that the inclusion of low-pressure homogenisation in a controlled fashion could be used to improve the sensory characteristics and consumer acceptability of cheese relative to un-treated cheeses. A pre-processing treatment was developed wherein raw milk was homogenised (at 0, 5 or 10 MPa pressure), incubated for 1 h at 37ºC and either batch pasteurised (63ºC for 30 min) or high-temperature short-time pasteurised (75ºC for 15 s). Control milks were treated similarly, but bypassed the homogenisation step. Cheddar and Emmental cheese were produced from the pre-treated milk and ripened according to respective ripening schemes. Reduced-fat Emmental cheese (20% fat) was also produced, using both homogenisation pressure of 10 MPa in the pre-treatment and with a control milk. Biochemical analyses showed higher moisture and NaCl levels in homogenised milk cheeses, while free fatty acids (FFA) were in some cases twice that of the control cheeses. The difference in amount of FFA can be attributed primarily to the lipolysis experienced in the pre-treatment. No differences were found in levels of bacteria in cheeses in any study. Confocal laser scanning microscopy (CLSM) showed a greater distribution of smaller homogenised fat globules throughout the cheese matrix. Sensory characteristics of the cheeses produced using the pre-treatment and Emmental cheesemaking were evaluated by descriptive sensory profiling using trained panels. Cheeses produced with homogenisation were rated higher for taste intensity and salty taste and were smoother, fattier and crumblier, lacked characteristic eyes and were less yellow and more colour consistent than those without homogenisation in the pre-treatment. Dynamic sensory characteristics were evaluated with the temporal dominance of sensations (TDS) method, where the evolution of the most dominant sensory attribute during mastication was followed. Homogenisation caused a drastic change in the texture during mastication. The control cheese was dominantly elastic at the start and then crumbly until swallowing while the homogenised milk cheese was experienced as crumbly-fatty-smooth. Evaluation of consumer responses and market positioning was carried out by projective mapping (PM), where respondents (n = 46) positioned the control and homogenised full-fat and reduced-fat test cheeses with six commercial cheeses. Similar cheeses were positioned closer together and different cheeses further apart. Participants were free to use their own criteria to position the cheeses. The reduced-fat homogenised cheese was rated as higher as more pleasant and positioned apart from similar commercial Emmental cheeses and the control cheeses. The full-fat homogenised milk cheese, ripened for 3 months, was rated higher for liking and positioned far from the other test cheeses, close to Gruyère-type (ripened for 7 months) and Gouda (ripened for 8 months) cheeses, described with words like full , tasty , melt-in-mouth and creamy . The positioning close to longer-ripened cheeses suggests a potential saving, through attainment of similar characteristics in a shorter ripening time. The effect of information on the expectations and perceptions of consumers was investigated. Participants (n = 229) completed a questionnaire with demographic and psychographic measures and were divided into four balanced groups based on age, sex, food neophobia and food technology neophobia. Each group received the same cheese sample and a different description, either, traditional , new-type , technology or cheese . Participants rated expected/actual pleasantness and purchase intent, as well as the suitability of descriptive words. Descriptions affected expected purchase intent, where the new-type group reported highest purchase intent, while no effect was found on expected pleasantness. Communication of novelty and technology raised the purchase intent of those with low food neophobia and low food technology neophobia, respectively. The pre-treatment is a viable and valuable addition prior to cheesemaking. Improvements in certain key sensory taste and texture attributes were seen, as well as improved positioning and consumer responses, indicating market potential and potential reduction in ripening costs. Although care should be taken in communication of both novelty and the technological nature of the process, the potential advantages of the pre-treatment are clear. Not only could the pre-treatment be applied prior to the manufacture of other types of cheeses, but potentially to other types of ripened-dairy products
  • Siljamäki, Pia (Helsingin yliopisto, 2014)
    Staphylococcus epidermidis (SE) is an opportunistic pathogen capable of infecting humans and animals. It is a frequent cause of hospital-acquired infections in humans and of intramammary infections (IMIs) in dairy cows. The protein expression profiles and the genomes of three SE strains, one associated with bovine mastitis (PM221), one representing a commensal/low-virulent human strain (ATCC12228 isolated from a healthy human host) and the third being a virulent human strain (RP62A isolated from a catheter-associated sepsis) were compared. At the genome and total proteome levels, the bovine strain PM221 was found to be more similar to ATCC12228 than to RP62A. The strain-specific differences found between PM221 and ATCC12228 are believed to have roles in adaptation and virulence. These findings may explain why ATCC12228 was able to cause persistent mastitis in an experimental infection study albeit with a milder clinical outcome than encountered with PM221. These findings strengthen the hypothesis that humans could represent an important source of SE-mediated infections in dairy cows. The exoproteome analysis revealed that the exoproteome of PM221 resembled more that of RP62A than the exoproteome of ATCC12228. The major part of the identified exoproteins was predicted to be cytoplasmic, indicating that these proteins might be surface-displayed moonlighting proteins or that they had been embedded in membrane vesicles. PM221 and RP62A were shown to be more efficient in non-classical protein secretion. These findings may explain the higher virulence capacity of PM221 than that of ATCC12228. Our studies also indicated that PM221 uses less aggressive strategies than RP62A when establishing an infection in vivo. Phenotypic tests indicated that SE may use several strategies to improve its adaptation to different environments; the virulent bovine and human strains (PM221 and RP62A) were able to use biofilm formation for colonization and adaptation, and PM221 and ATCC12228 could downregulate their tricarboxylic acid cycle activity and increase the formation of small colony variants to improve bacterial survival during the stationary phase. In addition, the bovine PM221 strain may possess an advantage since it has higher tributyrin activity in the milk environment, whereas the human strains may benefit from their higher urease and/or catalase activities helping these strains to survive in their ecological niches and indwelling medical devices. In conclusion, the present studies demonstrate that the SE species may exploit diverse strategies involving specific changes in their late stationary phase metabolism as well as in protein export strategies accompanied by increased production of certain enzymes in order to ensure better adaptation and/or successful infection.
  • Kuusinen, Nea (Helsingin yliopisto, 2014)
    Surface albedo refers to the fraction of solar irradiance that is reflected by a surface. Accurate characterisation of the albedo of various land cover types is required for evaluating the energy exchange between the Earth s surface and the atmosphere. The optical and structural properties of a surface determine its albedo. Boreal forest albedo can vary due to factors such as tree species composition, forest structure, understorey vegetation composition, and seasonal changes in vegetation and snow cover. The aim of this study was to characterise typical albedos of Finnish forests dominated by different tree species, evaluate the seasonal variation in forest albedo, and to estimate the effects of structural forest variables and understorey composition on forest albedo or spectral reflectance. To achieve these aims, forest albedo was measured in-situ using pyranometers, estimated from satellite data and calculated using a forest albedo model. Unmixing methods were used to estimate forest albedo from coarse spatial resolution MODIS albedo retrievals and understorey spectral reflectance from Landsat observations. Mature or middle aged pine, spruce and broadleaved deciduous (mainly birch) forests had distinctly different albedos in both summer and winter. Coniferous forest albedo was lower and showed less seasonal variation than albedo in open areas or broadleaved deciduous forests. Albedo of pine was somewhat higher than that of spruce. Snow cover on the ground and canopy increased forest albedo. Young stands with an assumedly high proportion of deciduous species in the under- and overstorey were characterised by a higher albedo than the mature coniferous forests. The high albedo at early succession rapidly decreased as the forest matured. The forest floor was typically covered by green understorey vegetation with rather low albedo, which decreased the influence of a changing canopy cover or leaf area index (LAI) on forest albedo. The spectral reflectances of the understorey varied with site fertility and forest age.
  • Zhang, Yijing (Helsingin yliopisto, 2014)
    With far-reaching impacts of economic globalization, the internationalization process of the forest industry has been accelerated. Particularly since the 1990s, internationalization has progressed intensively through industry consolidation and production relocation. Within the wood and wood products sector, for example, world inward foreign direct investment (FDI) stocks increased from 20 billion US dollars in 1990 to more than 120 billion US dollars in 2011. Forest industry firms have geographically shifted their operations from traditional production bases in developed countries to emerging Asian and Latin American countries. The share of FDI flows into developing and transition economies has grown from 18.8% in 1990 - 1992 to 73% in 2009 - 2011. The internationalization process of the forest industry has induced multifaceted concerns from economic, strategic, and environmental perspectives at both country and corporate level. However, the actual consequences of such expansion are still largely unknown. It is therefore important to study the current status of the forest industry s internationalization process and to explore drivers and goals of this process. The theoretical background of this thesis is mainly based on the internationalization and FDI theories portrayed in international business literature. A systematic literature review, a qualitative case study and cross-sectional regression analysis have been applied methodologically. China is used as an example in two of the empirical studies, as it is the most attractive FDI destination in the global forest sector. This thesis conceptually depicts a framework of the systematic internationalization process of the forest industry. Empirically, three themes are identified as focal topics; namely corporate financial performance, corporate sustainability, and corporate entry mode choice. Managerial implications derived from this thesis indicate that (1) firms could aim for either internationalized or domestic-oriented operational strategies to pursue higher financial performance; (2) firms should implement social and environmental assessment to maintain sustainable overseas operations; and (3) firms ought to accumulate operational experience and familiarity with local culture before investing in a wholly owned subsidiary. In general, this thesis concludes that internationalization is a dynamic process of pursuing sustainable development to tackle physical forest resource constraints, socio-economic challenges, and corporate operational risks at the global operations scale. Topics related to the analysis of new innovative products, collaboration with supporting industries, and the consideration of sustainability as corporate core competitive advantages are worthy of future research aiming to analyze the further internationalization process of the forest industry.
  • Hoornstra, Douwe (Helsingin yliopisto, 2014)
    Bacillus cereus is a ubiquitous food poisoning bacterium, but only producers of the emetic toxin, cereulide can be life threatening. Therefore a fast and reliable method is needed for identifying strains that produce the toxin. In this thesis the previously developed sperm bioassay was refined into a tool for the detection of microbially produced mitochondria toxic substances. This refined tool, Sperm Combi Assay effectively detected not only cereulide, but also other mitochondrial toxic substances and yielded data for classifying the substances into 3 different classes. The cereulide class (includes also valinomycin and enniatin) was characterised by a spotted mitochondrial sheath when stained with the membrane potential responsive dye JC-1. The gramicidin class (includes nigericin, calcimycin, monensin, narasin, salinomycin and antimycin A) was characterised by gradual fading of the dye during loss of the membrane potential. The ionomycin type (includes staurosporine, oligomycin) was characterised by loss of sperm motility with no changes of the mitochondrial membrane potential. The novel Sperm Combi Assay was put to the test to clarify the cause of illness connected to consumption of food supplements (seaweed capsules). The Sperm Combi Assay revealed highly toxic contaminants in the capsules. The test revealed that the poisonous substance was in the seaweed inside the capsule, not involving the capsule shell. The currently available official methods had failed to disclose this information. The test further helped to identify bacteria responsible for producing the toxic substances in the seaweed. The effects shown by the Sperm Combi Assay were similar to the gramicidin type of response. Next the Sperm Combi Assay was used to track potential sources of cereulide producers in food. We found cereulide producing B. cereus from potato tuber originating from the consumer market and had not undergone industrial treatment. We describe in this thesis how the initial plate count procedure can be improved to recognise the cereulide producing B. cereus already on a plate. Cereulide producers have been reported in industrial foods and rice by many researchers. Our report appears to be the first one where potato crops were screened for cereulide producers by methods that were effective for detecting cereulide producing B. cereus. This development is important because the potato is a major food crop in the European diet. Not only potatoes but many other plants such as trees in the forest contain cereulide producing B. cereus as an endophyte. The forest industry makes food packaging material from wood pulp. To trace the behaviour cereulide producing B. cereus contained in wood, we used the same protocol which was useful for finding the cereulide producing B. cereus in potato. We found that, of the spores of cereulide producing B. cereus, ca. 5 % may be retained in the paper product. The Sperm Combi Assay was used to track the distribution of cereulide, potentially released into the pulp slurry by cereulide producing B. cereus. We found that ca. 10 % present in the pulp slurry was recovered in the paper product. We measured if cereulide contained in paper could migrate from the paper product into packaged food and found almost all cereulide leached into simulated fatty foods, but less than the detection limit into warm or cold drinks. Food waste contains valuable nutrients and should be recycled into agricultural production. The question arises about the fate of cereulide producing B. cereus contained in food. We studied the behaviour of cereulide producing B. cereus spores throughout four seasons in an industrial scale biowaste processing plant that involved a heating step of 24 min at 137 °C followed by anaerobic digestion. We traced cereulide producing B. cereus in the digested biowaste with the same culture methods and the Sperm Combi Assay which proved operative in potato and with non culture based methods (PCR). We showed that the liquid biofertilizer contained ca. 1000 total B. cereus per ml of which 10 % were cesB (indicator of cereulide synthase) positive. This persistence of cereulide producing B. cereus under harsh conditions demonstrates that it is not possible to eradicate cereulide producers from the food cycle. Instead, we have to learn to design our food processing protocols so as to not give cereulide producers a chance to accumulate the cereulide toxin in foods. In this study we used porcine sperm cells as a tool to detect cereulide (their sensitivity is high), with an EC100 (equal to lethal dose) 0.3 ng ml-1. The question is if sperm sensitivity to cereulide reflects the sensitivity of other kinds of cells and tissues, for instance in the human body. We assessed this question by comparing the sensitivity of primary cells, cell lines and pancreatic islets towards cereulide. We found that the EC100 values for loss of mitochondrial membrane potential (24 h exposure) to human peripheral blood mononuclear cells and keratinocytes, porcine kidney epithelial cells and murine fibroblasts and pancreatic islets (containing the beta cells, which produce insulin) ranged from 0.4 to 2 ng of cereulide per ml. The results show that different kinds of cells were similarly vulnerable to mitochondrial toxicity by cereulide. These results strongly indicate that the Sperm Combi Assay is useful not only for tracking cereulide in foods and environmental samples, but can also be useful in assessing human health risk by materials contaminated by cereulide. We also showed that the external concentration of potassium (300µM increased to 850-950 µM) influences the speed of the potassium efflux, measured in HaCaT and PBMC cells. We introduced this Sperm Combi Assay to deal with a bacterium and/or toxin that can hardly be eliminated in foods. This assay is a fast and reliable method to detect mitochondria toxic substances as well as the reliable plating method for screening the producing organisms. Because it is not possible to eliminate cereulide producing B. cereus, it should be kept under control by avoiding conditions permissive for cereulide production.
  • Jaber, Emad (Helsingin yliopisto, 2014)
    Little information is available concerning the interaction of Heterobasidion annosum with the roots of herbaceous angiosperm plants. We investigated the infec¬tion biology of H. annosum during challenge with the angi¬osperm model Arabidopsis and monitored the host response after exposure to various hormone elicitors, chemicals (chitin, glucan and chitosan) and fungal species. This necrotrophic pathogen of conifer trees was able to infect the Col-8 (Columbia) ecotype of Arabidopsis in laboratory inoculation experiments. The germi¬nated H. annosum spores had appressorium-like penetra¬tion structures that attached to the surface of the Arabidopsis roots. The subsequent invasive fungal growth led to the disin¬tegration of the vascular region of the root tissues. The progres¬sion of root rot symptoms in Arabidopsis was similar to the infection development that was previously documented in Scots pine seedlings. Analysis of defensin gene expression in response to various biotic and chemical treatments in Scots pine compared with Arabidopsis suggested functional differences in their regulation in the two studied hosts. In parallel to the above study, the expression patterns of other closely related proteins, Scots pine antimicrobial proteins (Sp-AMPs) and the structure and function of the encoded proteins were investigated. The Sp-AMPs exhibited increased levels of expression specifically when challenged with H. annosum, consistent with a function in conifer tree defenses. The Sp-AMPs were up-regulated after treatment with salicylic acid (SA) and with ethylene (ET). The Sp-AMPs possessed antifungal activity against H. annosum and caused morphological changes in its hyphae and spores. The Sp-AMPs directly bind soluble and insoluble β-(1,3)-glucans specifically and with high affinity. Furthermore, the addition of exogenous glucan is associated with increased levels of Sp-AMP expression in the conifer tree. It was concluded from Homology modeling and sequence comparisons that Sp-AMPs belong to a new family of antimicrobial proteins (PR-19) that are likely to act by binding the glucans, which are a major component of fungal cell walls. To evaluate the potential of Sp-AMP as a molecular marker for resistance tree breeding, we developed transgenic tobacco plants expressing the Sp-AMP gene. A bioassay of transgenic tobacco (Nicotiana tabacum L. cv. SR1) plants over-expressing Sp-AMP2 challenged with the necrotrophic tobacco pathogen Botrytis cinerea was further investigated. The necrotic lesions caused by B. cinerea on the non-transgenic tobacco leaves were severe and larger than those lesions formed on the transgenic line. The results suggest that Scots pine pathogenesis-related protein 19 (PR-19) confers increased tolerance against Botrytis cineria in transgenic tobacco. This study provided insight concerning the initial molecular characterization of the expression and regulation of this protein family. The potential utility of the Sp-AMP genes as resistance markers in the conifer tree H. annosum pathosystem merits further investigation.
  • Häkkinen, Mari (Helsingin yliopisto, 2014)
    Utilisation of non-edible, renewable lignocellulosic biomass for the production of second generation biofuels and chemicals is hindered especially by the high price of enzymes needed for biomass degradation. Filamentous fungi are natural producers of enzymes active against plant cell wall polymers. Especially the ascomycota fungus Trichoderma reesei is widely utilised in the industry for the production of cellulases and hemicellulases. However, the efficiency of enzyme production needs to be further improved in order to ensure economical production of biobased products. Several environmental factors affect protein production by filamentous fungi. Cellulase and hemicellulase genes of T. reesei are activated by inducer molecules derived from different substrates. The need for cooperation of different hydrolytic enzymes for the total degradation of plant cell wall material has led to coordinated expression of these genes. However, the extent and timing of induction can vary between different genes and especially the hemicellulase genes aredifferentially induced by various substrates. The direct regulation of cellulase and hemicellulase genes by transcriptional regulators has been widely studied and several activators and repressors of these genes have been characterized in detail. However, little is still known concerning the exact regulatory pathways and mechanisms utilised by the fungus for the accurate timing and composition of the hydrolytic enzymes produced. In this study, a genome-wide transcriptional analysis of T. reesei gene expression at different ambient pH conditions was conducted in order to identify genes affected by extracellular pH. The role of a T. reesei orthologue for the characterized pH regulator, PacC, in the expression of cellulase and hemicellulase genes was also studied. An extensive induction experiment together with transcriptional profiling was then utilised to study the effects of several different substrates on the expression of genes encoding carbohydrate active enzymes (CAZy). In addition, transcriptomics data was utilised for the identification of novel candidate regulators affecting cellulase and xylanase production by T. reesei. Transcriptional profiling identified pH as an important determinant of T. reesei gene expression. Ambient pH was also found to affect the expression of several cellulase and hemicellulase genes and more information on the role of a PacC orthologue in the expression of cellulase and hemicellulase genes was gained. A profiling study utilising different substrates as inducers together with a thorough annotation of the T. reesei CAZy genes revealed the expression patterns of novel candidate genes possibly involved in the degradation of different types of cellulosic and hemicellulosic substrates. In addition, a phylogenetic analysis indicated that functional diversification of the carbohydrate active enzymes of T. reesei is a rather common phenomenon and is reflected in the differential regulation of the corresponding genes. A transcription factor gene named ace3 was identified from the profiling data and was shown to be essential for cellulase production and for the expression of cellulase genes. Over-expression of ace3 led to improved production of cellulase and xylanase activities. Several other candidate regulators were also identified as interesting subjects for more detailed studies. Overall, the use of genome-wide methods increased understanding concerning the genome organisation of T. reesei and its possible evolutionary benefits, and enabled identification of co-regulated genomic regions possibly involved in enzyme production.