Biomassahydrolysaattien puhdistus uutolla

Lignoselluloosasta koostuvasta biomassasta valmistetaan hydrolysoimalla sokereita, jotka jatkojalostetaan fermentoimalla bioetanoliksi. Bioetanolia käytetään fossiilisten polttoaineiden korvaajana esimerkiksi ajoneuvoissa. Bioetanolin valmistuksessa pyritään mahdollisimman hyvään saantoon, jotta sen valmistus olisi taloudellisesti kannattavaa. Hydrolyysin aikana syntyy sokerien lisäksi orgaanisia happoja, furaanin johdannaisia sekä fenolisia yhdisteitä. Yleisimpiä syntyviä yhdisteitä ovat muun muassa etikkahappo, furfuraali ja hydroksimetyylifurfuraali. Nämä yhdisteet haittaavat sokerien fermentointiprosessia ja pienentävät etanolin saantoa. Fermentointiprosessia haittaavien yhdisteiden poistoon hydrolysaattiliuoksesta voidaan käyttää esimerkiksi haihdutusta, membraanierotusta, adsorptiota, saostusta, sekä uuttoa. Tämän työn tarkoituksena oli tutkia leikkaussekoittimen soveltuvuutta biomassahydrolysaatin epäpuhtauksien erotukseen. Lisäksi kirjallisuusosassa on esitetty hydrolysointiprosessissa syntyviä haitta-aineita ja niiden erotusmenetelmiä.

Recovery of biomass-derived valuable compounds using chromatographic and membrane separations

Utilization of biomass-based raw materials for the production of chemicals and materials is gaining an increasing interest. Due to the complex nature of biomass, a major challenge in its refining is the development of efficient fractionation and purification processes. Preparative chromatography and membrane filtration are selective, energy-efficient separation techniques which offer a great potential for biorefinery applications. Both of these techniques have been widely studied. On the other hand, only few process concepts that combine the two methods have been presented in the literature. The aim of this thesis was to find the possible synergetic effects provided by combining chromatographic and membrane separations, with a particular interest in biorefinery separation processes. Such knowledge could be used in the development of new, more efficient separation processes for isolating valuable compounds from complex feed solutions that are typical for the biorefinery environment. Separation techniques can be combined in various ways, from simple sequential coupling arrangements to fully-integrated hybrid processes. In this work, different types of combined separation processes as well as conventional chromatographic separation processes were studied for separating small molecules such as sugars and acids from biomass hydrolysates and spent pulping liquors. The combination of chromatographic and membrane separation was found capable of recovering high-purity products from complex solutions. For example, hydroxy acids of black liquor were successfully recovered using a novel multistep process based on ultrafiltration and size-exclusion chromatography. Unlike any other separation process earlier suggested for this challenging separation task, the new process concept does not require acidification pretreatment, and thus it could be more readily integrated into a pulp-mill biorefinery. In addition to the combined separation processes, steady-state recycling chromatography, which has earlier been studied for small-scale separations of high-value compounds only, was found a promising process alternative for biorefinery applications. In comparison to conventional batch chromatography, recycling chromatography provided higher product purity, increased the production rate and reduced the chemical consumption in the separation of monosaccharides from biomass hydrolysates. In addition, a significant further improvement in the process performance was obtained when a membrane filtration unit was integrated with recycling chromatography. In the light of the results of this work, separation processes based on combining membrane and chromatographic separations could be effectively applied for different biorefinery applications. The main challenge remains in the development of inexpensive separation materials which are resistant towards harsh process conditions and fouling.

Physiological response of vicia faba to prohexadione-calcium under saline conditions

Changes in the activities of oxidative enzymes (indole acetic acid oxidase, peroxidase and catalase), endogenous hormones (gibberellic acid (GA3), indole acetic acid (IAA), abscisic acid (ABA) and cytokinins (AsZeatin), photosynthetic pigments (chlorophyll a, chlorophyll b and carotenoids), total carbohydrates, total soluble sugars, amino acid proline and vegetative growth parameters were used as indicators to explain the physiological role of the growth retardant prohexadione-calcium on Vicia faba seedlings 40 days after sowing under salinity stress for 30 days. The obtained results show that soaking faba bean seeds prior to sowing at different concentrations of prohexadione-calcium (0, 10, 20 and 30 ppm) significantly increased the activities of indole acetic acid oxidase (IAA-oxidase) and peroxidase enzymes, but decreased the catalase enzyme activity as compared with their respective control. Application of prohexadione-Ca caused markedly decreases in the endogenous contents of gibberellins and indole acetic acid (IAA) but increased the levels of natural growth inhibitor abscisic acid (ABA) and cytokinins in the shoots of faba bean seedlings. All the prohexadione-Ca concentrations increased the contents of amino acid proline, photosynthetic pigments (chlorophyll a, chlorophyll b and carotenoids), total carbohydrates and total soluble sugars in faba bean seedlings grown under salt stress. Application of prohexadione-Ca decreased significantly seedling height and shoot fresh weight but significantly increased shoot dry weight.

Morphological and physiological alterations induced by lactofen in soybean leaves are reduced with nitric oxide

Lactofen is a diphenylether herbicide recommended to control broad-leaved weeds in soybean (Glycine max) fields and its mechanism of action is the inhibition of protoporphyrinogen-IX oxidase (Protox), which acts in the chlorophyll biosynthesis. This inhibition results in an accumulation of protoporphyrin-IX, which leads to the production of reactive oxygen species (ROS) that cause oxidative stress. Consequently, spots, wrinkling and leaf burn may occur, resulting in a transitory crop growth interruption. However, nitric oxide (NO) acts as an antioxidant in direct ROS scavenging. Thus, the aim of this work was to verify, through phytometric and biochemical evaluations, the protective effect of NO in soybean plants treated with the herbicide lactofen. Soybean plants were pre-treated with different levels of sodium nitroprusside (SNP), a NO-donor substance, and then sprayed with 168 g a.i. ha-1 lactofen. Pre-treatment with SNP was beneficial because NO decreased the injury symptoms caused by lactofen in young leaflets and kept low the soluble sugar levels. Nevertheless, NO caused slower plant growth, which indicates that further studies are needed in order to elucidate the action mechanisms of NO in signaling the stress caused by lactofen in soybean crop.

Pressure filtration characteristics of enzymatically hydrolyzed biomass suspensions

Enzymatic hydrolysis of lignocellulosic polymers is likely to become one of the key technologies enabling industrial production of liquid biofuels and chemicals from lignocellulosic biomass. Certain types of enzymes are able to hydrolyze cellulose and hemicellulose polymers to shorter units and finally to sugar monomers. These monomeric sugars are environmentally acceptable carbon sources for the production of liquid biofuels, such as bioethanol, and other chemicals, such as organic acids. Liquid biofuels in particular have been shown to contribute to the reduction of net emissions of greenhouse gases. The solid residue of enzymatic hydrolysis is composed mainly of lignin and partially degraded fibers, while the liquid phase contains the produced sugars. It is usually necessary to separate these two phases at some point after the hydrolysis stage. Pressure filtration is an efficient technique for this separation. Solid-liquid separation of biomass suspensions is difficult, because biomass solids are able to retain high amounts of water, which cannot be readily liberated by mechanical separation techniques. Most importantly, the filter cakes formed from biomaterials are compressible, which ultimately means that the separation may not be much improved by increasing the filtration pressure. The use of filter aids can therefore facilitate the filtration significantly. On the other hand, the upstream process conditions have a major influence on the filtration process. This thesis investigates how enzymatic hydrolysis and related process conditions affect the filtration properties of a cardboard suspension. The experimental work consists of pressure filtration and characterization of hydrolysates. The study provides novel information about both issues, as the relationship between enzymatic hydrolysis conditions and subsequent filtration properties has so far not been considered in academic studies. The results of the work reveal that the final degree of hydrolysis is an important factor in the filtration stage. High hydrolysis yield generally increases the average specific cake resistance. Mixing during the hydrolysis stage resulted in undefined changes in the physical properties of the solid residue, causing a high filtration resistance when the mixing intensity was high. Theoretical processing of the mixing data led to an interesting observation: the average specific cake resistance was observed to be linearly proportional to the mixer shear stress. Another finding worth attention is that the size distributions of the solids did not change very dramatically during enzymatic hydrolysis. There was an observable size reduction during the first couple of hours, but after that the size reduction was minimal. Similarly, the size distribution of the suspended solids remained almost constant when the hydrolyzed suspension was subjected to intensive mixing. It was also found that the average specific cake resistance was successfully reduced by the use of filter aids. This reduction depended on the method of how the filter aids were applied. In order to obtain high filtration capacity, it is recommended to use the body feed mode, i.e. to mix the filter aid with the slurry prior to filtration. Regarding the quality of the filtrate, precoat filtration was observed to produce a clear filtrate with negligible suspended solids content, while the body feed filtrates were turbid, irrespective of which type of filter aid was used.

Characterization of Leaf-Type Ferredoxin-NADP+ Oxidoreductase (FNR) Isoforms in Arabidopsis thaliana

Life on earth is based on sunlight, which is captured in chemical form by photosynthetic reactions. In the chloroplasts of plants, light reactions of photosynthesis take place at thylakoid membranes, whereas carbon assimilation reactions occur in the soluble stroma. The products of linear electron transfer (LET), highly-energetic ATP molecules, and reducing power in the form of NADPH molecules, are further used in the fixation of inorganic CO2 molecules into organic sugars. Ferredoxin-NADP+ oxidoreductase (FNR) catalyzes the last of the light reactions by transferring electrons from ferredoxin (FD) to NADP+. In addition to LET, FNR has been suggested to play a role in cyclic electron transfer (CET), which produces ATP without the accumulation of reducing equivalents. CET is proposed to occur via two putative routes, the PGR5- route and the NDH-route. In this thesis, the leaf-type FNR (LFNR) isoforms LFNR1 and LFNR2 of a model organism, Arabidopsis thaliana, were characterized. The physiological roles of LFNRs were investigated using single and double mutant plants. The viability of the single mutants indicates functionality of both isoforms, with neither appearing to play a specific role in CET. The more severe phenotype of low-temperature adapted fnr2 plants compared to both wild-type (WT) and fnr1 plants suggests a specific role for LFNR2 under unfavorable growth conditions. The more severe phenotype of the fnr1 x fnr2 (F1 generation) plants compared to single mutants reflects down-regulated photosynthetic capacity, whereas slightly higher excitation pressure indicates mild over-excitation of electron transfer chain (ETC). However, induction of CET and various photoprotective mechanisms enable adaptation of fnr1 x fnr2 plants to scarcity of LFNR. The fnr1 fnr2 plants (F2 generation), without detectable levels of LFNR, were viable only under heterotrophic conditions. Moreover, drought stress induced acceleration of the rate of P700 + re-reduction in darkness was accompanied by a concomitant up-regulation of the PGR5-route specific components, PGR5 and PGRL1, demonstrating the induction of CET via the PGR5-route. The up-regulation of relative transcriptional expression of the FD1 gene indicates that the FD1 isoform may have a specific function in CET, while no such role could be defined for either of the LFNR isoforms. Both the membrane-bound and soluble LFNR1 and LFNR2 each appear as two distinct spots after 2D-PAGE with different isoelectric points (pIs), indicating the existence of post-translational modifications (PTMs) which do not determine the membrane attachment of LFNR. The possibility of phosphorylation and glycosylation PTMs were excluded, but all four LFNR forms were shown to contain acetylated lysine residues as well as alternative N-termini. N-terminal acetylation was shown to shift the pI of both LFNRs to be more acidic. In addition, all four LFNR forms were demonstrated to interact both with FD1 and FD2 in vitro

Reserve substances and storage of Cyathea delgadii Sternb. spores

The spores used were collected from a population of trees of Cyathea delgadii Sternb. growing in a gallery forest at the Reserva Biológica e Estação Experimental de Moji Guaçu, São Paulo state, Brazil (22°18’S and 47°11’W). The germination of spores of Cyathea delgadii decreases with time when kept in closed bottles under storage at 4°C in darkness. Germination is still very high after storage for one year. Spores stored for three years do not germinate. The results also show a decrease in soluble proteins and an increase in starch after several months storage.

Intracellular and intercellular compartmentation of carbohydrate metabolism in leaves of temperate gramineae

This review considers the importance of compartmentation in the regulation of carbohydrate metabolism in leaves. We draw particular attention to the role of the vacuole as a site for storage of soluble sugars based on sucrose, and discuss briefly their characteristic metabolism. We also point out inconsistencies between the observed properties of vacuoles and the behaviour in vitro of the enzymes of fructan biosynthesis that do not support the hypothesis that the vacuole is the site of synthesis as well as of storage. We also consider compartmentation of carbohydrate metabolism between different cell types, using mainly our studies on leaves of temperate C3 gramineae. Here we present evidence of significant differences in carbon metabolism between epidermis, mesophyll, bundle sheath and vasculature based upon both single-cell sampling and immunolocalisation. The implications of these differences for the control of metabolism in leaves are discussed.

Reproductive biology of the protandrous Ferdinandusa speciosa Pohl (Rubiaceae) in Southeastern Brazil

A study of the floral biology and the breeding system of Ferdinandusa speciosa Pohl (Rubiaceae) was carried out from March to September 1996 in Uberlândia, MG, central Brazil. This species is a shrub or small tree that occurs in swampy edges of gallery forests. The two studied populations flowered somewhat asynchronously from March to July. The tubular flowers are red, approximately 4.7 cm long and last for two days. They are protandrous and the pollen is available one day before the stigma becomes receptive. The beginning of anthesis and the opening of the stigmatic lips occur at dusk. The nectar is secreted during both the male and the female phases, with concentration of sugars greater in the male phase. The flowers are pollinated by two hummingbird species, Chlorostilbon aureoventris and Phaethornis pretrei. Ferdinandusa speciosa is a self-compatible, non-apomictic species, with low fruit production under natural conditions in the populations studied. No differences were found between fruit set of self- and cross-pollinated flowers, nor in the pollen tube growth rate in the pistils of these flowers. The seeds formed by cross-pollination are larger, heavier and show a higher percentage of germination than those formed by self-pollination, which indicates inbreeding depression. This result suggests that, although the species is self-compatible, cross-pollination may be advantageous.

Diurnal variations of non-structural carbohydrates in vegetative tissues of Melinis minutiflora, Echinolaena inflexa and Lolium multiflorum (Poaceae)

Echinolaena inflexa (Poir.) Chase is a native C3 grass species with high biomass production and abundance in the cerrado comparable to Melinis minutiflora Beauv., a C4 forage grass of African origin, widespread in the cerrado, displacing some native herbaceous species. These species accumulate mainly starch, differently of Lolium multiflorum Lam. ssp. italicum Beck cv. Lema (ryegrass), a C3 temperate forage grass that accumulates fructans as the major storage carbohydrate in the vegetative tissues. In the present study, we analyzed diurnal variations in the content and composition of non-structural carbohydrates (NSC) in the aerial vegetative parts of the three species. Results showed similarity in water-soluble carbohydrates (WSC) and starch contents between Melinis minutiflora and Echinolaena inflexa, with the exotic grass accumulating higher amounts of NSC. However, the levels of sucrose, glucose and fructose were 10 fold higher in Lolium multiflorum, that accumulates fructan as the main storage carbohydrate. Although the contents of WSC and starch were similar in the tropical species E. inflexa and M. minutiflora, their diurnal variations were different and could be related to starch metabolism, especially in M. minutiflora.

Germination of spores and growth of gametophytes and sporophytes of Rumohra adiantiformis (Forst.) Ching (Dryopteridaceae) after spore cryogenic storage

Rumohra adiantiformis (Forst.) Ching is a fern (Dryopteridaceae) used in floral arrangements. Spores sterilized in 15% (v/v) solution of commercial sodium hypochlorite for 10 minutes and unsterilized spores were plunged in liquid nitrogen and held for 15 minutes and for 90 days. After the cryogenic treatments, spores were taken out of liquid nitrogen and rapidly thawed out in a water bath or slowly at room temperature and were cultured in Mohr's mineral solution as modified by Dyer, kept at 25 ± 2 ºC and a 16-hours photoperiod. Statistical differences were not observed in the germination of unsterilized spores immersed or not immersed in liquid nitrogen, but when the spores were previously sterilized, a severe inhibition of germination was observed in cryopreserved spores. Faster mean germination time was observed for unsterilized spores cryopreserved in liquid nitrogen for 15 minutes. The germination of spores stored in liquid nitrogen for 90 days reached the maximum percentage after 12 days, while control spores reached their maximum percentage after 16 days. Levels of soluble sugars did not vary among treatments in gametophytes cultivated for 10 weeks after spore inoculation. The number of fronds and the length of the longest frond on sporophytes did not differ statistically among treatments. The relative growth rate of sporophytes grown from cryopreserved and control spores were not statistically different among treatments. Spores of R. adiantiformis immersed in liquid nitrogen for 15 minutes apparently produced phenotypically normal plants.

Anatomy, ultrastructure and secretion of Hibiscus pernambucensis Arruda (Malvaceae) extrafloral nectary

This paper reports on the extrafloral nectary (EFN) of Hibiscus pernambucensis, a native shrub species occurring in mangrove and restinga along Brazil's coastline. EFNs occur as furrows with a protuberant border on the abaxial surface veins of the leaf blade. Each nectary consists of numerous secretory multicellular trichomes, epidermal cells in palisade-like arrangements and non-vascularized parenchyma tissue. Nectar secretion is prolonged, since secretion starts in very young leaves and remains up to completely expanded leaves. Reduced sugars, lipids, and proteins were histochemically detected in all the nectary cells; phenolic substances were detected in the vacuoles of the epidermal palisade cells and in some secretory trichome cells. The secretory cells that constitute the body of trichomes have large nuclei, dense cytoplasm with numerous mitochondria, dictyosomes, scattered lipid droplets and plastids with different inclusions: protein, lipid droplets or starch grains; vacuoles with different sizes have membranous material, phenolic and lipophilic substances. The palisade cells show thick periclinal walls, reduced cytoplasm with voluminous lipid drops and developed vacuoles. The nectary parenchyma cells contain abundant plasmodesmata and cytoplasm with scattered lipid droplets, mitochondria, plastids with starch grains and endoplasmic reticulum. Mucilage idioblasts are common in the inner nectary parenchyma. Protoderm and ground meristem participate in the formation of EFN. Our data indicate that all nectary regions are involved in nectar production and secretion, constituting a functional unit. Longevity of the extrafloral nectaries is likely associated with the presence of mucilage idioblasts, which increases the capacity of the nectary parenchyma to store water.

Flowering and fruiting of Aulonemia aristulata: a gynomonoecious woody bamboo species from Atlantic Forest in Brazil

Aulonemia aristulata (Döll) McClure is a lignified bamboo species endemic to Brazil. This species occurs in southeastern forests and can reach high density at forest edges, dominating the understory of canopy-disturbed forest patches. The goal of this study was to describe the flowering period, floral biology, fruiting and seedling recruitment of A. aristulata in natural conditions in two areas located in a segment of the Atlantic Forest. Data on the morphology of the synflorescences and florets, timing and sequence of the anthesis events and floral visitors were recorded. Natural pollinators (open pollination or control) as well as spontaneous self-pollination were also checked. Pollen viability was estimated using the acetocarmine technique. Aulonemia aristulata is monocarpic (semelparous) with gregarious flowering. All culms in both studied areas blossomed and fruited between August and November 2007, dying subsequently between December 2007 and April 2008. Two types of synflorescences and flowers were observed: terminal with bisexual and protandric florets, with the anthesis lasting for 3-4 days; and axillary, with morphologically bisexual, but functionally female, florets and anthesis lasting for 3-4 days. The latter were also observed in the rhizome of plants whose aerial portion had been removed. The presence of axillary synflorescences with pistillate flowers is described here for the first time in Aulonemia species. Moreover, this is the first report of gynomonoecy in woody bamboo. Fruiting from bisexual florets under natural conditions (35%) was superior to that obtained from bagged synflorescences (11.5%). Fruiting from functional female florets was around 20%. Pollen viability was on the average of 90%. The results suggest that Aulonemia aristulata is anemophilous. The massive bamboo seedling recruitment observed after dieback with the ability to colonize open areas could promote the regeneration of Aulonemia aristulata.

Diurnal changes in storage carbohydrate metabolism in cotyledons of the tropical tree Hymenaea courbaril L. (Leguminosae)

The cotyledons of Hymenaea courbaril store large amounts of xyloglucan, a cell wall polysaccharide that is believed to serve as storage for the period of seedling establishment. During storage mobilisation, xyloglucan seems to be degraded by a continuous process that starts right after radicle protrusion and follows up to the establishment of photosynthesis. Here we show evidence that events related to the hydrolases activities and production (α-xylosidase, β-galactosidase, β-glucosidase and xyloglucan endo-β-transglucosilase) as well as auxin, showed changes that follow the diurnal cycle. The period of higher hydrolases activities was between 6pm and 6am, which is out of phase with photosynthesis. Among the enzymes, α-xilosidase seems to be more important than β-glucosidase and β-galactosidase in the xyloglucan disassembling mechanism. Likewise, the sugars related with sucrose metabolism followed the rhythm of the hydrolases, but starch levels were shown to be practically constant. A high level of auxin was observed during the night, what is compatible with the hypothesis that this hormone would be one of the regulators of the whole process. The probable biological meaning of the existence of such a complex control mechanism during storage mobilisation is likely to be related to a remarkably high level of efficiency of carbon usage by the growing seedling of Hymenaea courbaril, allowing the establishment of very vigorous seedlings in the tropical forest.

Inflammatory and anti-inflammatory effects of soybean agglutinin

Soybean agglutinin (SBA) lectin, a protein present in raw soybean meals, can bind to and be extensively endocytosed by intestinal epithelial cells, being nutritionally toxic for most animals. In the present study we show that SBA (5-200 µg/cavity) injected into different cavities of rats induced a typical inflammatory response characterized by dose-dependent exudation and neutrophil migration 4 h after injection. This effect was blocked by pretreatment with glucocorticoid (0.5 mg/kg) or by co-injection of N-acetyl-galactosamine (100 x [M] lectin), but not of other sugars (100 x [M] lectin), suggesting an inflammatory response related to the lectin activity. Neutrophil accumulation was not dependent on a direct effect of SBA on the macrophage population since the effect was not altered when the number of peritoneal cells was increased or decreased in vivo. On the other hand, SBA showed chemotactic activity for human neutrophils in vitro. A slight increase in mononuclear cells was observed 48 h after ip injection of SBA. Phenotypic analysis of these cells showed an increase in the CD4+/CD8- lymphocyte population that returned to control levels after 15 days, suggesting the development of an immune response. SBA-stimulated macrophages presented an increase in the expression of CD11/CD18 surface molecules and showed some characteristics of activated cells. After intravenous administration, SBA increased the number of circulating neutrophils and inhibited in a dose-dependent manner the neutrophil migration induced by ip injection of carrageenan into peritoneal cavities. The co-injection of N-acetyl-galactosamine or mannose, but not glucose or fucose, inhibited these effects. The data indicate that soybean lectin is able to induce a local inflammatory reaction but has an anti-inflammatory effect when present in circulating blood