Diversitet och tillväxtfrämjande egenskaper hos Stenotrophomonas-endofyter isolerade från Calliandra calothyrsus Meisn. rotknölar

The bacterial genus Stenotrophomonas comprises 12 species. They are widely found throughout the environment and particularly S. maltophilia, S. rhizophila and S. pavanii are closely associated with plants. Strains of the most common Stenotrophomonas species, S. maltophilia, promote plant growth and health, degrade natural and man-made pollutants and produce biomolecules of biotechnological and economical value. Many S. maltophilia –strains are also multidrug resistant and can act as opportunistic human pathogens. During an INCO-project (1998-2002) rhizobia were collected from root nodules of the tropical leguminous tree Calliandra calothyrsus Meisn. from several countries in Central America, Africa and New Caledonia. The strains were identified by the N2-group (Helsinki university) and some strains turned out to be members of the genus Stenotrophomonas. Several Stenotrophomonas strains induced white tumor- or nodule-like structures on Calliandra?s roots in plant experiments. The strains could, besides from root nodules, also be isolated from surface sterilized roots and stems. The purpose of my work was to investigate if the Stenotrophomonas strains i) belong to a new Stenotrophomonas species, ii) have the same origin, iii) if there are other differences than colony morphology between phase variations of the same strain, iv) have plant growth-promoting (PGP) activity or other advantageous effects on plants, and v) like rhizobia have ability to induce root nodule formation. The genetic diversity and clustering of the Stenotrophomonas strains were analyzed with AFLP fingerprinting to get indications about their geographical origin. Differences in enzymatic properties and ability to use different carbon and energy sources were tested between the two phases of each strain with commercial API tests for bacterial identification. The ability to infect root hairs and induce root nodule formation was investigated both using plant tests with the host plant Calliandra and PCR amplification of nodA and nodC genes for nodulation. The PGP activity of the strains was tested in vitro mainly with plate methods. The impact on growth, nitrogen content and nodulation in vivo was investigated through greenhouse experiments with the legumes Phaseolus vulgaris and Galega orientalis. Both the genetic and phenotypic diversity among the Stenotrophomonas strains was small, which proposes that they have the same origin. The strains brought about changes on the root hairs of Calliandra and they also increased the amount of root hairs. However, no root nodules were detected. The strains produced IAA, protease and lipase in vitro. They also showed plant a growth-promoting effect on G. orientalis, both alone and together with R. galegae HAMBI 540, and also activated nodulation among efficient rhizobia on P. vulgaris in greenhouse. It requires further research to get a better picture about the mechanisms behind the positive effects. The results in this thesis, however, confirm earlier studies concerning Stenotrophomonas positive impact on plants.

Emerging bioenergy business at Finnish independent industrial sawmills

Finnish forest industry is in the middle of a radical change. Deepening recession and the falling demand of woodworking industry´s traditional products have forced also sawmilling industry to find new and more fertile solutions to improve their operational preconditions. In recent years, the role of bioenergy production has often been highlighted as a part of sawmills´ business repertoire. Sawmilling produces naturally a lot of by-products (e.g. bark, sawdust, chips) which could be exploited more effectively in energy production, and this would bring more incomes or maybe even create new business opportunities for sawmills. Production of bioenergy is also supported by government´s climate and energy policies favouring renewable energy sources, public financial subsidies, and soaring prices of fossil fuels. Also the decreasing production of domestic pulp and paper industry releases a fair amount of sawmills´ by-products for other uses. However, bioenergy production as a part of sawmills´ by-product utilization has been so far researched very little from a managerial point of view. The purpose of this study was to explore the relative significance of the main bioenergy-related processes, resources and factors at Finnish independent industrial sawmills including partnerships, cooperation, customers relationships and investments, and also the future perspectives of bioenergy business at these sawmills with the help of two resource-based approaches (resource-based view, natural-resource-based view). Data of the study comprised of secondary data (e.g. literature), and primary data which was attracted from interviews directed to sawmill managers (or equivalent persons in charge of decisions regarding bioenergy production at sawmill). While a literature review and the Delphi method with two questionnaires were utilized as the methods of the study. According to the results of the study, the most significant processes related to the value chain of bioenergy business are connected to raw material availability and procurement, and customer relationships management. In addition to raw material and services, the most significant resources included factory and machinery, personnel, collaboration, and geographic location. Long-term cooperation deals were clearly valued as the most significant form of collaboration, and especially in processes connected to raw material procurement. Study results also revealed that factors related to demand, subsidies and prices had highest importance in connection with sawmills´ future bioenergy business. However, majority of the respondents required that certain preconditions connected to the above-mentioned factors should be fulfilled before they will continue their bioenergy-related investments. Generally, the answers showed a wide divergence of opinions among the respondents which may refer to sawmills´ different emphases and expectations concerning bioenergy. In other words, bioenergy is still perceived as a quite novel and risky area of business at Finnish independent industrial sawmills. These results indicate that the massive expansion of bioenergy business at private sawmills in Finland is not a self-evident truth. The blocking barriers seem to be connected mainly to demand of bioenergy and money. Respondents´ answers disseminated a growing dissatisfaction towards the policies of authorities, which don´t treat equally sawmill-based bioenergy compared to other forms of bioenergy. This proposition was boiled down in a sawmill manager´s comment: “There is a lot of bioenergy available, if they just want to make use of it.” It seems that the positive effects of government´s policies favouring the renewables are not taking effect at private sawmills. However, as there anyway seems to be a lot of potential connected to emerging bioenergy business at Finnish independent industrial sawmills, there is also a clear need for more profound future studies over this topic.

Effects of dietary phosphorus and calcium-to-phosphorus ratio on calcium and bone metabolism in healthy 20- to 43-year-old Finnish women

Dietary habits have changed during the past decades towards an increasing consumption of processed foods, which has notably increased not only total dietary phosphorus (P) intake, but also intake of P from phosphate additives. While the intake of calcium (Ca) in many Western countries remains below recommended levels (800 mg/d), the usual daily P intake in a typical Western diet exceeds by 2- to 3-fold the dietary guidelines (600 mg/d). The effects of high P intake in healthy humans have been investigated seldom. In this thesis healthy 20- to 43-year-old women were studied. In the first controlled study (n = 14), we examined the effects of P doses, and in a cross-sectional study (n = 147) the associations of habitual P intakes with Ca and bone metabolism. In this same cross-sectional study, we also investigated whether differences exist between dietary P originating from natural P sources and phosphate additives. The second controlled study (n = 12) investigated whether by increasing the Ca intake, the effects of a high P intake could be reduced. The associations of habitual dietary calcium-to-phosphorus ratios (Ca:P ratio) with Ca and bone metabolism were determined in a cross-sectional study design (n = 147). In the controlled study, the oral intake of P doses (495, 745, 1245 and 1995 mg/d) with a low Ca intake (250 mg/d) increased serum parathyroid hormone (S-PTH) concentration in a dose-dependent manner. In addition, the highest P dose decreased serum ionized calcium (S-iCa) concentration and bone formation and increased bone resorption. In the second controlled study with a dietary P intake of 1850 mg/d, by increasing the Ca intake from 480 mg/d to 1080 mg/d and then to 1680 mg/d, the S-PTH concentration decreased, the S-iCa concentration increased and bone resorption decreased dose-dependently. However, not even the highest Ca intake could counteract the effect of high dietary P on bone formation, as indicated by unchanged bone formation activity. In the cross-sectional studies, a higher habitual dietary P intake (>1650 mg/d) was associated with lower S-iCa and higher S-PTH concentrations. The consumption of phosphate additive-containing foods was associated with a higher S-PTH concentration. Moreover, habitual low dietary Ca:P ratios (≤0.50, molar ratio) were associated with higher S-PTH concentrations and 24-h urinary Ca excretions, suggesting that low dietary Ca:P ratios may interfere with homeostasis of Ca metabolism and increase bone resorption. In summary, excessive dietary P intake in healthy Finnish women seems to be detrimental to Ca and bone metabolism, especially when dietary Ca intake is low. The results indicate that by increasing dietary Ca intake to the recommended level, the negative effects of high P intake could be diminished, but not totally prevented. These findings imply that phosphate additives may be more harmful than natural P. Thus, reduction of an excessively high dietary P intake is also beneficial for healthy individuals.

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

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

Transcriptome and proteome analysis of xylose-metabolising Saccharomyces cerevisiae

Increasing concern about global climate warming has accelerated research into renewable energy sources that could replace fossil petroleum-based fuels and materials. Bioethanol production from cellulosic biomass by fermentation with baker s yeast Saccharomyces cerevisiae is one of the most studied areas in this field. The focus has been on metabolic engineering of S. cerevisiae for utilisation of the pentose sugars, in particular D-xylose that is abundant in the hemicellulose fraction of biomass. Introduction of a heterologous xylose-utilisation pathway into S. cerevisiae enables xylose fermentation, but ethanol yield and productivity do not reach the theoretical level. In the present study, transcription, proteome and metabolic flux analyses of recombinant xylose-utilising S. cerevisiae expressing the genes encoding xylose reductase (XR) and xylitol dehydrogenase (XDH) from Pichia stipitis and the endogenous xylulokinase were carried out to characterise the global cellular responses to metabolism of xylose. The aim of these studies was to find novel ways to engineer cells for improved xylose fermentation. The analyses were carried out from cells grown on xylose and glucose both in batch and chemostat cultures. A particularly interesting observation was that several proteins had post-translationally modified forms with different abundance in cells grown on xylose and glucose. Hexokinase 2, glucokinase and both enolase isoenzymes 1 and 2 were phosphorylated differently on the two different carbon sources studied. This suggests that phosphorylation of glycolytic enzymes may be a yet poorly understood means to modulate their activity or function. The results also showed that metabolism of xylose affected the gene expression and abundance of proteins in pathways leading to acetyl-CoA synthesis and altered the metabolic fluxes in these pathways. Additionally, the analyses showed increased expression and abundance of several other genes and proteins involved in cellular redox reactions (e.g. aldo-ketoreductase Gcy1p and 6-phosphogluconate dehydrogenase) in cells grown on xylose. Metabolic flux analysis indicated increased NADPH-generating flux through the oxidative part of the pentose phosphate pathway in cells grown on xylose. The most importantly, results indicated that xylose was not able to repress to the same extent as glucose the genes of the tricarboxylic acid and glyoxylate cycles, gluconeogenesis and some other genes involved in the metabolism of respiratory carbon sources. This suggests that xylose is not recognised as a fully fermentative carbon source by the recombinant S. cerevisiae that may be one of the major reasons for the suboptimal fermentation of xylose. The regulatory network for carbon source recognition and catabolite repression is complex and its functions are only partly known. Consequently, multiple genetic modifications and also random approaches would probably be required if these pathways were to be modified for further improvement of xylose fermentation by recombinant S. cerevisiae strains.

Biomassan energia-arvon nostaminen sekundäärilämmöllä kuivaamalla

Viime aikoina ilmastonmuutos, fossiilisten polttoaineiden väheneminen ja niiden hinnan nousu ovat lisänneet merkittävästi maailmanlaajuista kiinnostusta uusiutuviin energiavaroihin. Suomessa uusiutuvien energialähteiden käytössä on jo pitkään panostettu metsäteollisuuden sivutuotevirtana tuottamaan puuperäiseen biomassaan, jota metsäteollisuus käyttää energiantuotantoonsa. Metsäteollisuuden jätevesien käsittelyssä syntyy erilaisia lietteitä, jotka joko uusiokäytetään tai hävitetään polttamalla tai sijoittamalla kaatopaikalle. Erityisesti biolietteiden uusiokäyttö on hankalaa ja kaatopaikkasijoitus tulevaisuudessa mahdotonta tai ainakin kustannuksiltaan kohtuutonta. Käytännössä liete hävitetään polttamalla ja kuivaamalla siitä tulee polttoaine. Lietteiden energiakäyttö on järkevin tapa hävittää jäteliete. Lietteiden korkean vesipitoisuuden vuoksi ne tulee kuitenkin kuivata ennen polttoa. Lietteen kuivaaminen sekundäärienergiavirralla eli metsäteollisuusprosesseissa sivutuotteena muodostuvalla ns. hukkalämmöllä lisää lietteen poltosta saatavaa energiamäärää ja korvaa fossiilisten polttoaineiden käyttöä. Tutkimuksen tavoitteena oli selvittää lietteen kuivaukseen optimaalisin kuoren ja lietteen seossuhde eri kuivausparametrejä vaihdellen. Kokeellinen työ aloitettiin rakentamalla energiatekniikan koehalliin laboratoriokokoluokan kiintopetikuivuri, jossa kuivumista tutkittiin puhaltamalla polttoainepedin läpi lämmitettyä ilmaa. Kuivattavina polttoaineina olivat kuoren ja lietteen seos tai pelkkä kuori ja liete erilaisilla massoilla ja erilaisilla prosenttisilla suhteilla ja erilaisissa lämpötiloissa. Kuivumiskäyrien määritys perustui massanmuutokseen. Koelaitteessa olivat anturit lämpötilan mittausta varten, jotta lämpötila saatiin säädettyä ja seurattua kokeen edellyttämällä tavalla. Lämpötilat ja painonmuutokset tallentuivat koetta tehdessä tietokoneelle. Kuivauskokeet osoittivat, että liete-kuori seos kuivuu hyvin kiintopedissä kun lietteen massaosuus seoksessa on korkeintaan 50 %. Lietteen massaosuuden ollessa tätä suurempi kuivaaminen ei enää ole tehokasta, mikä johtuu luultavasti ilman suuresta kanavoitumisesta kuivauspedissä. Kuorta kuivatessa lämpötilan nosto 50 °C:stä 70 °C:een oli huomattavasti tehokkaampaa kuin 70 °C:stä 90 °C:een, ajallisesti ero oli noin kaksinkertainen.

Reconstructions of past climates from documentary and natural sources in Finland since the 18th century

In this paper both documentary and natural proxy data have been used to improve the accuracy of palaeoclimatic knowledge in Finland since the 18th century. Early meteorological observations from Turku (1748-1800) were analyzed first as a potential source of climate variability. The reliability of the calculated mean temperatures was evaluated by comparing them with those of contemporary temperature records from Stockholm, St. Petersburg and Uppsala. The resulting monthly, seasonal and yearly mean temperatures from 1748 to 1800 were compared with the present day mean values (1961-1990): the comparison suggests that the winters of the period 1749-1800 were 0.8 ºC colder than today, while the summers were 0.4 ºC warmer. Over the same period, springs were 0.9 ºC and autumns 0.1 ºC colder than today. Despite their uncertainties when compared with modern meteorological data, early temperature measurements offer direct and daily information about the weather for all months of the year, in contrast with other proxies. Secondly, early meteorological observations from Tornio (1737-1749) and Ylitornio (1792-1838) were used to study the temporal behaviour of the climate-tree growth relationship during the past three centuries in northern Finland. Analyses showed that the correlations between ring widths and mid-summer (July) temperatures did not vary significantly as a function of time. Early (June) and late summer (August) mean temperatures were secondary to mid-summer temperatures in controlling the radial growth. According the dataset used, there was no clear signature of temporally reduced sensitivity of Scots pine ring widths to mid-summer temperatures over the periods of early and modern meteorological observations. Thirdly, plant phenological data with tree-rings from south-west Finland since 1750 were examined as a palaeoclimate indicator. The information from the fragmentary, partly overlapping, partly nonsystematically biased plant phenological records of 14 different phenomena were combined into one continuous time series of phenological indices. The indices were found to be reliable indicators of the February to June temperature variations. In contrast, there was no correlation between the phenological indices and the precipitation data. Moreover, the correlations between the studied tree-rings and spring temperatures varied as a function of time and hence, their use in palaeoclimate reconstruction is questionable. The use of present tree-ring datasets for palaeoclimate purposes may become possible after the application of more sophisticated calibration methods. Climate variability since the 18th century is perhaps best seen in the fourth paper study of the multiproxy spring temperature reconstruction of south-west Finland. With the help of transfer functions, an attempt has been made to utilize both documentary and natural proxies. The reconstruction was verified with statistics showing a high degree of validity between the reconstructed and observed temperatures. According to the proxies and modern meteorological observations from Turku, springs have become warmer and have featured a warming trend since around the 1850s. Over the period of 1750 to around 1850, springs featured larger multidecadal low-frequency variability, as well as a smaller range of annual temperature variations. The coldest springtimes occurred around the 1840s and 1850s and the first decade of the 19th century. Particularly warm periods occurred in the 1760s, 1790s, 1820s, 1930s, 1970s and from 1987 onwards, although in this period cold springs occurred, such as the springs of 1994 and 1996. On the basis of the available material, long-term temperature changes have been related to changes in the atmospheric circulation, such as the North Atlantic Oscillation (February-June).