Synthese cyclischer Peptide und von Kohlenhydraten abgeleitete Katalysatoren zur enantioselektiven Darstellung von Cyanhydrinen

Der Suche nach neuen Wirkstoffen für den chemischen Pflanzenschutz kommt insbesondere vor dem Hintergrund der steigenden Weltbevölkerung und weniger zur Verfügung stehenden kulturfähigen Ackerflächen eine stetig wachsende Bedeutung zu. Ziel dieser Arbeit war die Synthese von cyclischen Peptiden und Depsipeptiden, die aufgrund ihrer biologischen Aktivität als potentielle Insektizide für den chemischen Pflanzenschutz in Frage kommen. Darüber hinaus sollten von Kohlenhydraten abgeleitete Katalysatoren zur enantioselektiven Cyanhydrinsynthese entwickelt werden, um einen leichten Zugang zu den Bausteinen der Depsipeptide zu ermöglichen. Als vielversprechender Naturstoff mit insektiziden Eigenschaften gilt das cyclische Pentapeptid Cycloaspeptid E, dessen Totalsynthese in 10 Stufen mit einer Gesamtausbeute von 25% erreicht wurde, sodass die Verbindung für biologische Tests bereitgestellt werden konnte. Zusätzlich gelang die Kristallisation der Verbindung, was eine Röntgenstrukturanalyse ermöglichte. Ein Derivat von Cycloaspeptid E sollte 2-Aminonicotinsäure anstelle von Anthranilsäure enthalten. Die Synthese dieser Verbindung wurde auf drei Wegen versucht. Dabei zeigte sich, dass es bei einer zur Totalsynthese des Naturstoffs analogen Strategie zur quantitativen Bildung eines Diketopiperazins kommt. Auf den anderen Routen ließ sich entweder ein Kupplungsschritt nicht realisieren, oder die Verbindung erwies sich unter den gewählten Bedingungen als instabil. Die Darstellung eines 2-Aminonicotinsäure-Derivats von Cycloaspeptid E bleibt daher weiterhin ein ungelöstes Problem, das weiterer Forschung bedarf. Verticilid A1 ist ein cyclisches Depsipeptid, das aufgrund seiner Bindungsfähigkeit an den Ryanodinrezeptor von Insekten, als Leitstruktur für die Suche nach neuen Insektiziden von Interesse ist. Um zu untersuchen, wie wichtig die Esterbindungen im Molekül für die biologische Aktivität sind, sollte das entsprechende Amid-Derivat und das Cyclodepsipeptid mit nur zwei statt vier Esterbindungen hergestellt werden. Hierbei zeigte sich, dass eine zur Darstellung von Verticilid A1 analoge Syntheseroute zu einer ausgeprägten Epimerisierung führt. Eine lineare Synthese der Derivate endet in der Bildung des Diketopiperazins. Weiterhin wurden zwei neue, zueinander pseudoenantiomere Vanadium(IV)-Katalysatoren auf Basis von D-Glucose einerseits und L-Xylose andererseits dargestellt. Diese lassen sich in fünf bzw. 14 Stufen synthetisieren und liefern in der enantioselektiven Katalyse von Mandelsäurenitril Enantiomerenüberschüsse von 89% bzw. 91% bei hohen Ausbeuten. Zusammenfassend lässt sich feststellen, dass im Rahmen dieser Arbeit die Totalsynthese von Cycloaspeptid E erfolgreich durchgeführt wurde, und die Syntheseversuche von weiteren cyclischen Peptiden wichtige Erkenntnisse für weitere Synthesen lieferten. Mit den beiden hergestellten Vanadium(IV)-Komplexen wurden zwei potente, auf Kohlenhydraten basierende Katalysatoren für die enantioselektive Synthese von Cyanhydrinen entwickelt.

Starch distribution in pear tree organs in relation to training systems, rootstocks and fruit quality

Starch is the main form in which plants store carbohydrates reserves, both in terms of amounts and distribution among different plant species. Carbohydrates are direct products of photosynthetic activity, and it is well know that yield efficiency and production are directly correlated to the amount of carbohydrates synthesized and how these are distributed among vegetative and reproductive organs. Nowadays, in pear trees, due to the modernization of orchards, through the introduction of new rootstocks and the development of new training systems, the understanding and the development of new approaches regarding the distribution and storage of carbohydrates, are required. The objective of this research work was to study the behavior of carbohydrate reserves, mainly starch, in different pear tree organs and tissues: i.e., fruits, leaves, woody organs, roots and flower buds, at different physiological stages during the season. Starch in fruit is accumulated at early stages, and reached a maximum concentration during the middle phase of fruit development; after that, its degradation begins with a rise in soluble carbohydrates. Moreover, relationships between fruit starch degradation and different fruit traits, soluble sugars and organic acids were established. In woody organs and roots, an interconversion between starch and soluble carbohydrates was observed during the dormancy period that confirms its main function in supporting the growth and development of new tissues during the following spring. Factors as training systems, rootstocks, types of bearing wood, and their position on the canopy, influenced the concentrations of starch and soluble carbohydrates at different sampling dates. Also, environmental conditions and cultural practices must be considered to better explain these results. Thus, a deeper understanding of the dynamics of carbohydrates reserves within the plant could provide relevant information to improve several management practices to increase crop yield efficiency.

1st and 2nd Generation Ethanol from Biomass Crops

In chapter 1 and 2 calcium hydroxide as impregnation agent before steam explosion of sugarcane bagasse and switchgrass, respectively, was compared with auto-hydrolysis, assessing the effects on enzymatic hydrolysis and simultaneous saccharification and fermentation (SSF) at high solid concentration of pretreated solid fraction. In addition, anaerobic digestion of pretreated liquid fraction was carried out, in order to appraise the effectiveness of calcium hydroxide before steam explosion in a more comprehensive way. In As water is an expensive input in both cultivation of biomass crops and subsequent pretreatment, Chapter 3 addressed the effects of variable soil moisture on biomass growth and composition of biomass sorghum. Moreover, the effect of water stress was related to the characteristics of stem juice for 1st generation ethanol and structural carbohydrates for 2nd generation ethanol. In the frame of chapter 1, calcium hydroxide was proven to be a suitable catalyst for sugarcane bagasse before steam explosion, in order to enhance fibre deconstruction. In chapter 2, effect of calcium hydroxide on switchgrass showed a great potential when ethanol was focused, whereas acid addition produced higher methane yield. Regarding chapter 3, during crop cycle the amount of cellulose, hemicellulose and AIL changed causing a decrease of 2G ethanol amount. Biomass physical and chemical properties involved a lower glucose yield and concentration at the end of enzymatic hydrolysis and, consequently, a lower 2G ethanol concentration at the end of simultaneous saccharification and fermentation, proving that there is strong relationship between structure, chemical composition, and fermentable sugar yield. The significantly higher concentration of ethanol at the early crop stage could be an important incentive to consider biomass sorghum as second crop in the season, to be introduced into some agricultural systems, potentially benefiting farmers and, above all, avoiding the exacerbation of the debate about fuel vs food crops.

Isolierung von Essigsäure-, Propionsäure- und Buttersäure-bildenden Bakterien aus Biogasanlagen

In der vorliegenden Arbeit wurden Essigsäure-, Propionsäure und Buttersäure-bildende Bakterien aus einer thermophilen und drei mesophilen Biogasanlagen sowie aus zwei Hochdruck-Biogas-Laborfermentern isoliert. Die Fermenter waren mit dem nachwachsenden Rohstoff Maissilage, teilweise mit Rinder- oder Schweinegülle und weiteren festen Inputstoffen gefüttert. Für die Isolierung von Säure-bildenden Bakterien wurde ein Mineralsalzmedium verwendet, welchem als Kohlenstoffquelle Na-DL-Laktat, Succinat, Ethanol, Glycerin, Glucose oder eine Aminosäuremischung (Alanin, Serin, Threonin, Glutaminsäure, Methionin und Cystein) hinzugefügt wurde. Hierbei handelt es sich um Substrate, welche beim anaeroben Abbau während der Hydrolyse oder der primären Gärung entstehen können. Die erhaltenen Isolate waren in der Lage, aus diesen Substraten Essigsäure, Propionsäure oder Buttersäure zu bilden. Insgesamt wurden aus den beprobten Anlagen 49 Isolate gewonnen, welche zu den Phyla Firmicutes, Tenericutes oder Thermotogae gehörten. Mit Hilfe von 16S rDNA-Sequenzen konnten die meisten Isolate als Clostridium sporosphaeroides, Defluviitoga tunisiensis und Dendrosporobacter sp. identifiziert werden. Die Bildung von Essigsäure, Propionsäure oder Buttersäure wurde in Kulturen von Isolaten festgestellt, welche als folgende Arten identifiziert wurden: Bacillus thermoamylovorans, Clostridium aminovalericum, Clostridium cochlearium/Clostridium tetani, Clostridium sporosphaeroides, Dendrosporobacter sp., Proteiniborus sp., Selenomonas bovis und Tepidanaerobacter sp. Zwei Isolate, verwandt mit Thermoanaerobacterium thermosaccharolyticum, konnten Buttersäure und Milchsäure bilden. In Kulturen von Defluviitoga tunisiensis wurde Essigsäurebildung festgestellt. Ein Vergleich der 16S rDNA-Sequenzen mit Datenbanken und die Ergebnisse der PCR-Amplifikationen mit Isolat-spezifischen Primerpaaren ergaben zusätzlich Hinweise, dass es sich bei einigen Isolaten um neue Arten handeln könnte (z. B. Stamm Tepidanaerobacter sp. AS34, Stamm Proteiniborus sp. ASG1.4, Stamm Dendrosporobacter sp. LG2.4, Stamm Desulfotomaculum sp. EG2.4, Stamm Gallicola sp. SG1.4B und Stamm Acholeplasma sp. ASSH51). Durch die Entwicklung Isolat-spezifischer Primerpaare, abgeleitet von 16S rDNA-Sequenzen der Isolate oder Referenzstämmen, konnten die Isolate in Biogasanlagen detektiert und mittels qPCR quantifiziert werden (hauptsächlich im Bereich zwischen 1000 bis 100000000 Kopien der 16S rDNA/g BGA-Probe). Weiterhin konnten die Isolate mit Hilfe physiologischer Versuche charakterisiert und deren Rolle in der anaeroben Abbaukette diskutiert werden. Die Art Defluviitoga tunisiensis scheint eine große Bedeutung in Biogasanlagen zu spielen. Defluviitoga tunisiensis wurde am häufigsten in Untersuchungen im Rahmen der vorliegenden Arbeit isoliert und konnte auch mit Hilfe des entwickelten Primerpaares in hohen Abundanzen in den beprobten Biogasanlagen detektiert werden (10000 - 100000000 Kopien der 16S rDNA/g BGA-Probe). Die manuelle Annotation des Gesamtgenoms sowie die Substratverwertungsversuche haben gezeigt, dass Defluviitoga tunisiensis ein sehr breites Substratspektrum in der Verwertung von Kohlenhydraten besitzt und dadurch möglicherweise eine wichtige Rolle bei der Verwertung von Biomasse in Biogasanlagen einnimmt. Mit Hilfe der Ergebnisse der vorliegenden Arbeit konnten somit neue Einblicke in die zweite Stufe des anaeroben Abbaus, die Acidogenese, in Biogasanlagen gegeben werden. rn

Effects of a short-term overfeeding with fructose or glucose in healthy young males

Consumption of simple carbohydrates has markedly increased over the past decades, and may be involved in the increased prevalence in metabolic diseases. Whether an increased intake of fructose is specifically related to a dysregulation of glucose and lipid metabolism remains controversial. We therefore compared the effects of hypercaloric diets enriched with fructose (HFrD) or glucose (HGlcD) in healthy men. Eleven subjects were studied in a randomised order after 7 d of the following diets: (1) weight maintenance, control diet; (2) HFrD (3.5 g fructose/kg fat-free mass (ffm) per d, +35 % energy intake); (3) HGlcD (3.5 g glucose/kg ffm per d, +35 % energy intake). Fasting hepatic glucose output (HGO) was measured with 6,6-2H2-glucose. Intrahepatocellular lipids (IHCL) and intramyocellular lipids (IMCL) were measured by 1H magnetic resonance spectroscopy. Both fructose and glucose increased fasting VLDL-TAG (HFrD: +59 %, P < 0.05; HGlcD: +31 %, P = 0.11) and IHCL (HFrD: +52 %, P < 0.05; HGlcD: +58 %, P = 0.06). HGO increased after both diets (HFrD: +5 %, P < 0.05; HGlcD: +5 %, P = 0.05). No change was observed in fasting glycaemia, insulin and alanine aminotransferase concentrations. IMCL increased significantly only after the HGlcD (HFrD: +24 %, NS; HGlcD: +59 %, P < 0.05). IHCL and VLDL-TAG were not different between hypercaloric HFrD and HGlcD, but were increased compared to values observed with a weight maintenance diet. However, glucose led to a higher increase in IMCL than fructose.

Glycomic analysis of human mast cells, eosinophils and basophils

In allergic diseases such as asthma, eosinophils, basophils and mast cells, through release of preformed and newly generated mediators, granule proteins and cytokines, are recognized as key effector cells. While their surface protein phenotypes, mediator release profiles, ontogeny, cell trafficking and genomes have been generally explored and compared, there has yet to be any thorough analysis and comparison of their glycomes. Such studies are critical to understand the contribution of carbohydrates to the induction and regulation of allergic inflammatory responses and are now possible using improved technologies for detecting and characterizing cell-derived glycans. We thus report here the application of high-sensitivity mass spectrometric-based glycomics methodologies to the analysis of N-linked glycans derived from isolated populations of human mast cells, eosinophils and basophils. The samples were subjected to matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) screening analyses and MALDI-TOF/TOF sequencing studies. Results reveal substantive quantities of terminal N-acetylglucosamine containing structures in both the eosinophil and the basophil samples, whereas mast cells display greater relative quantities of sialylated terminal epitopes. For the first time, we characterize the cell surface glycan structures of principal allergic effector cells, which by interaction with glycan-binding proteins (e.g. lectins) have the possibility to dictate cellular functions, and might thus have important implications for the pathogenesis of inflammatory and allergic diseases.

Ramachandran-type Plots for Glycosidic Linkages: Examples from Molecular Dynamics Simulations using the Glycam06 Force Field

The goals of this article are to (1) provide further validation of the Glycam06 force field, specifically for its use in implicit solvent molecular dynamic (MD) simulations, and (2) to present the extension of G.N. Ramachandran's idea of plotting amino acid phi and psi angles to the glycosidic phi, psi, and omega angles formed between carbohydrates. As in traditional Ramachandran plots, these carbohydrate Ramachandran-type (carb-Rama) plots reveal the coupling between the glycosidic angles by displaying the allowed and disallowed conformational space. Considering two-bond glycosidic linkages, there are 18 possible conformational regions that can be defined by (α, ϕ, ψ) and (β, ϕ, ψ), whereas for three-bond linkages, there are 54 possible regions that can be defined by (α, ϕ, ψ, ω) and (β, ϕ, ψ, ω). Illustrating these ideas are molecular dynamic simulations on an implicitly hydrated oligosaccharide (700 ns) and its eight constituent disaccharides (50 ns/disaccharide). For each linkage, we compare and contrast the oligosaccharide and respective disaccharide carb-Rama plots, validate the simulations and the Glycam06 force field through comparison to experimental data, and discuss the general trends observed in the plots.

An Actor-Critic based controller for glucose regulation in type 1 diabetes

A novel adaptive approach for glucose control in individuals with type 1 diabetes under sensor-augmented pump therapy is proposed. The controller, is based on Actor-Critic (AC) learning and is inspired by the principles of reinforcement learning and optimal control theory. The main characteristics of the proposed controller are (i) simultaneous adjustment of both the insulin basal rate and the bolus dose, (ii) initialization based on clinical procedures, and (iii) real-time personalization. The effectiveness of the proposed algorithm in terms of glycemic control has been investigated in silico in adults, adolescents and children under open-loop and closed-loop approaches, using announced meals with uncertainties in the order of ±25% in the estimation of carbohydrates. The results show that glucose regulation is efficient in all three groups of patients, even with uncertainties in the level of carbohydrates in the meal. The percentages in the A+B zones of the Control Variability Grid Analysis (CVGA) were 100% for adults, and 93% for both adolescents and children. The AC based controller seems to be a promising approach for the automatic adjustment of insulin infusion in order to improve glycemic control. After optimization of the algorithm, the controller will be tested in a clinical trial.

Patterns of functional enzyme activity in fungus farming ambrosia beetles

Introduction In wood-dwelling fungus-farming weevils, the so-called ambrosia beetles (Curculionidae: Scolytinae and Platypodinae), wood in the excavated tunnels is used as a medium for cultivating fungi by the combined action of digging larvae (which create more space for the fungi to grow) and of adults sowing and pruning the fungus. The beetles are obligately dependent on the fungus that provides essential vitamins, amino acids and sterols. However, to what extent microbial enzymes support fungus farming in ambrosia beetles is unknown. Here we measure (i) 13 plant cell-wall degrading enzymes in the fungus garden microbial consortium of the ambrosia beetle Xyleborinus saxesenii, including its primary fungal symbionts, in three compartments of laboratory maintained nests, at different time points after gallery foundation and (ii) four specific enzymes that may be either insect or microbially derived in X. saxesenii adult and larval individuals. Results We discovered that the activity of cellulases in ambrosia fungus gardens is relatively small compared to the activities of other cellulolytic enzymes. Enzyme activity in all compartments of the garden was mainly directed towards hemicellulose carbohydrates such as xylan, glucomannan and callose. Hemicellulolytic enzyme activity within the brood chamber increased with gallery age, whereas irrespective of the age of the gallery, the highest overall enzyme activity were detected in the gallery dump material expelled by the beetles. Interestingly endo-β-1,3(4)-glucanase activity capable of callose degradation was identified in whole-body extracts of both larvae and adult X. saxesenii, whereas endo-β-1,4-xylanase activity was exclusively detected in larvae. Conclusion Similar to closely related fungi associated with bark beetles in phloem, the microbial symbionts of ambrosia beetles hardly degrade cellulose. Instead, their enzyme activity is directed mainly towards comparatively more easily accessible hemicellulose components of the ray-parenchyma cells in the wood xylem. Furthermore, the detection of xylanolytic enzymes exclusively in larvae (which feed on fungus colonized wood) and not in adults (which feed only on fungi) indicates that only larvae (pre-) digest plant cell wall structures. This implies that in X. saxesenii and likely also in many other ambrosia beetles, adults and larvae do not compete for the same food within their nests - in contrast, larvae increase colony fitness by facilitating enzymatic wood degradation and fungus cultivation.

An investigation of phenolic glycoside and condensed tannin homeostasis in Populus by salicyl alcohol feeding to cell cultures and by transgenic manipulation of the sucrose transporter, PTSUT4, in planta

Secondary metabolites play an important role in plant protection against biotic and abiotic stress. In Populus, phenolic glycosides (PGs) and condensed tannins (CTs) are two such groups of compounds derived from the common phenylpropanoid pathway. The basal levels and the inducibility of PGs and CTs depend on genetic as well as environmental factors, such as soil nitrogen (N) level. Carbohydrate allocation, transport and sink strength also affect PG and CT levels. A negative correlation between the levels of PGs and CTs was observed in several studies. However, the molecular mechanism underlying such relation is not known. We used a cell culture system to understand negative correlation of PGs and CTs. Under normal culture conditions, neither salicin nor higher-order PGs accumulated in cell cultures. Several factors, such as hormones, light, organelles and precursors were discussed in the context of aspen suspension cells’ inability to synthesize PGs. Salicin and its isomer, isosalicin, were detected in cell cultures fed with salicyl alcohol, salicylaldehyde and helicin. At higher levels (5 mM) of salicyl alcohol feeding, accumulation of salicins led to reduced CT production in the cells. Based on metabolic and gene expression data, the CT reduction in salicin-accumulating cells is partly a result of regulatory changes at the transcriptional level affecting carbon partitioning between growth processes, and phenylpropanoid CT biosynthesis. Based on molecular studies, the glycosyltransferases, GT1-2 and GT1-246, may function in glycosylation of simple phenolics, such as salicyl alcohol in cell cultures. The uptake of such glycosides into vacuole may be mediated to some extent by tonoplast localized multidrug-resistance associated protein transporters, PtMRP1 and PtMRP6. In Populus, sucrose is the common transported carbohydrate and its transport is possibly regulated by sucrose transporters (SUTs). SUTs are also capable of transporting simple PGs, such as salicin. Therefore, we characterized the SUT gene family in Populus and investigated, by transgenic analysis, the possible role of the most abundantly expressed member, PtSUT4, in PG-CT homeostasis using plants grown under varying nitrogen regimes. PtSUT4 transgenic plants were phenotypically similar to the wildtype plants except that the leaf area-to-stem volume ratio was higher for transgenic plants. In SUT4 transgenics, levels of non-structural carbohydrates, such as sucrose and starch, were altered in mature leaves. The levels of PGs and CTs were lower in green tissues of transgenic plants under N-replete, but were higher under N-depleted conditions, compared to the levels in wildtype plants. Based on our results, SUT4 partly regulates N-level dependent PG-CT homeostasis by differential carbohydrate allocation.

Kinetic characterization for pretreatment of timber varieties and switchgrass using diluted acid hydrolysis

In recent years, growing attention has been devoted to the use of lignocellulosic biomass as a feedstock to produce renewable carbohydrates as a source of energy products, including liquid alternatives to fossil fuels. The benefits of developing woody biomass to ethanol technology are to increase the long-term national energy security, reduce fossil energy consumption, lower greenhouse gas emissions, use renewable rather than depletable resources, and create local jobs. Currently, research is driven by the need to reduce the cost of biomass-ethanol production. One of the preferred methods is to thermochemically pretreat the biomass material and subsequently, enzymatically hydrolyze the pretreated material to fermentable sugars that can then be converted to ethanol using specialized microorganisms. The goals of pretreatment are to remove the hemicellulose fraction from other biomass components, reduce bioconversion time, enhance enzymatic conversion of the cellulose fraction, and, hopefully, obtain a higher ethanol yield. The primary goal of this research is to obtain kinetic detailed data for dilute acid hydrolysis for several timber species from the Upper Peninsula of Michigan and switchgrass. These results will be used to identify optimum reaction conditions to maximize production of fermentable sugars and minimize production of non-fermentable byproducts. The structural carbohydrate analysis of the biomass species used in this project was performed using the procedure proposed by National Renewable Energy Laboratory (NREL). Subsequently, dilute acid-catalyzed hydrolysis of biomass, including aspen, basswood, balsam, red maple, and switchgrass, was studied at various temperatures, acid concentrations, and particle sizes in a 1-L well-mixed batch reactor (Parr Instruments, ii Model 4571). 25 g of biomass and 500 mL of diluted acid solution were added into a 1-L glass liner, and then put into the reactor. During the experiment, 5 mL samples were taken starting at 100°C at 3 min intervals until reaching the targeted temperature (160, 175, or 190°C), followed by 4 samples after achieving the desired temperature. The collected samples were then cooled in an ice bath immediately to stop the reaction. The cooled samples were filtered using 0.2 μm MILLIPORE membrane filter to remove suspended solids. The filtered samples were then analyzed using High Performance Liquid Chromatography (HPLC) with a Bio-Rad Aminex HPX-87P column, and refractive index detection to measure monomeric and polymeric sugars plus degradation byproducts. A first order reaction model was assumed and the kinetic parameters such as activation energy and pre-exponential factor from Arrhenius equation were obtained from a match between the model and experimental data. The reaction temperature increases linearly after 40 minutes during experiments. Xylose and other sugars were formed from hemicellulose hydrolysis over this heat up period until a maximum concentration was reached at the time near when the targeted temperature was reached. However, negligible amount of xylose byproducts and small concentrations of other soluble sugars, such as mannose, arabinose, and galactose were detected during this initial heat up period. Very little cellulose hydrolysis yielding glucose was observed during the initial heat up period. On the other hand, later in the reaction during the constant temperature period xylose was degraded to furfural. Glucose production from cellulose was increased during this constant temperature period at later time points in the reaction. The kinetic coefficient governing the generation of xylose from hemicellulose and the generation of furfural from xylose presented a coherent dependence on both temperature and acid concentration. However, no effect was observed in the particle size. There were three types of biomass used in this project; hardwood (aspen, basswood, and red maple), softwood (balsam), and a herbaceous crop (switchgrass). The activation energies and the pre-exponential factors of the timber species and switchgrass were in a range of 49 - 180 kJ/mol and from 7.5x104 - 2.6x1020 min-1, respectively, for the xylose formation model. In addition, for xylose degradation, the activation energies and the preexponential factors ranged from 130 - 170 kJ/mol and from 6.8x1013 - 3.7x1017 min-1, respectively. The results compare favorably with the literature values given by Ranganathan et al, 1985. Overall, up to 92 % of the xylose was able to generate from the dilute acid hydrolysis in this project.

THE POTENTIAL OF INDUSTRIAL WASTE AND AGRICULTURAL FEEDSTOCK TOWARDS SUSTAINABLE BIOFUELS PRODUCTION: TECHNO-ECONOMIC AND ENVIRONMENTAL IMPACT PERSPECTIVES

This Ph.D. research is comprised of three major components; (i) Characterization study to analyze the composition of defatted corn syrup (DCS) from a dry corn mill facility (ii) Hydrolysis experiments to optimize the production of fermentable sugars and amino acid platform using DCS and (iii) Sustainability analyses. Analyses of DCS included total solids, ash content, total protein, amino acids, inorganic elements, starch, total carbohydrates, lignin, organic acids, glycerol, and presence of functional groups. Total solids content was 37.4% (± 0.4%) by weight, and the mass balance closure was 101%. Total carbohydrates [27% (± 5%) wt.] comprised of starch (5.6%), soluble monomer carbohydrates (12%) and non-starch carbohydrates (10%). Hemicellulose components (structural and non-structural) were; xylan (6%), xylose (1%), mannan (1%), mannose (0.4%), arabinan (1%), arabinose (0.4%), galatactan (3%) and galactose (0.4%). Based on the measured physical and chemical components, bio-chemical conversion route and subsequent fermentation to value added products was identified as promising. DCS has potential to serve as an important fermentation feedstock for bio-based chemicals production. In the sugar hydrolysis experiments, reaction parameters such as acid concentration and retention time were analyzed to determine the optimal conditions to maximize monomer sugar yields while keeping the inhibitors at minimum. Total fermentable sugars produced can reach approximately 86% of theoretical yield when subjected to dilute acid pretreatment (DAP). DAP followed by subsequent enzymatic hydrolysis was most effective for 0 wt% acid hydrolysate samples and least efficient towards 1 and 2 wt% acid hydrolysate samples. The best hydrolysis scheme DCS from an industry's point of view is standalone 60 minutes dilute acid hydrolysis at 2 wt% acid concentration. The combined effect of hydrolysis reaction time, temperature and ratio of enzyme to substrate ratio to develop hydrolysis process that optimizes the production of amino acids in DCS were studied. Four key hydrolysis pathways were investigated for the production of amino acids using DCS. The first hydrolysis pathway is the amino acid analysis using DAP. The second pathway is DAP of DCS followed by protein hydrolysis using proteases [Trypsin, Pronase E (Streptomyces griseus) and Protex 6L]. The third hydrolysis pathway investigated a standalone experiment using proteases (Trypsin, Pronase E, Protex 6L, and Alcalase) on the DCS without any pretreatment. The final pathway investigated the use of Accellerase 1500® and Protex 6L to simultaneously produce fermentable sugars and amino acids over a 24 hour hydrolysis reaction time. The 3 key objectives of the techno-economic analysis component of this PhD research included; (i) Development of a process design for the production of both the sugar and amino acid platforms with DAP using DCS (ii) A preliminary cost analysis to estimate the initial capital cost and operating cost of this facility (iii) A greenhouse gas analysis to understand the environmental impact of this facility. Using Aspen Plus®, a conceptual process design has been constructed. Finally, both Aspen Plus Economic Analyzer® and Simapro® sofware were employed to conduct the cost analysis as well as the carbon footprint emissions of this process facility respectively. Another section of my PhD research work focused on the life cycle assessment (LCA) of commonly used dairy feeds in the U.S. Greenhouse gas (GHG) emissions analysis was conducted for cultivation, harvesting, and production of common dairy feeds used for the production of dairy milk in the U.S. The goal was to determine the carbon footprint [grams CO2 equivalents (gCO2e)/kg of dry feed] in the U.S. on a regional basis, identify key inputs, and make recommendations for emissions reduction. The final section of my Ph.D. research work was an LCA of a single dairy feed mill located in Michigan, USA. The primary goal was to conduct a preliminary assessment of dairy feed mill operations and ultimately determine the GHG emissions for 1 kilogram of milled dairy feed.

Practical handling of AIO admixtures - Guidelines on Parenteral Nutrition, Chapter 10

All-in-one admixtures (AIO-admixtures) provide safe, effective and low-risk PN (parenteral nutrition) for practically all indications and applications. Water, energy (carbohydrates and lipids), amino acids, vitamins and trace elements are infused together with PN either as industrially-manufactured AIO admixtures provided as two- or three-chamber bags (shelf life usually more than 12 months) completed with electrolytes and micronutrients where appropriate or as individually compounded ready-to-use AIO admixtures (compounding, usually prepared by a pharmacy on either a daily or weekly basis and stored at 2-8 degrees C). Physico-chemical and microbial stability of an AIO admixture is essential for the safety and effectiveness of patient-specific PN, and its assurance requires specialist pharmaceutical knowledge. The stability should be documented for an application period of 24 (-48) hours. It is advisable to offer a limited selection of different PN regimes in each hospital. For reasons of drug and medication safety, PN admixtures prepared for individual patients must be correctly labelled and specifications for storage conditions must also be followed during transport. Monitoring is required where applicable. Micronutrients are usually administered separately to AIO admixtures. In case compatibility and stability have been well documented trace elements and/or combination preparations including water-soluble or water-soluble/fat soluble vitamin supplements can be added to PN admixtures under strict aseptic conditions. AIO admixtures are usually not used as vehicles for drugs (incompatibilities).

Mild and efficient hydrolysis of thioglycosides to glycosyl hemiacetals using N-iodosaccharin

A convenient methodology has been developed for the mild hydrolysis of thioglycosides to the corresponding hemiacetals using N-iodosaccharin without any requirement of co-activator. Most of the functional groups used for the protecting group manipulation of carbohydrates are remain unaffected under the reaction condition.

Immuno-detection of anthrose containing tetrasaccharide in the exosporium of Bacillus anthracis and Bacillus cereus strains

AIMS: Bacillus anthracis strains of various origins were analysed with the view to describe intrinsic and persistent structural components of the Bacillus collagen-like protein of anthracis glycoprotein associated anthrose containing tetrasaccharide in the exosporium. METHODS AND RESULTS: The tetrasaccharide consists of three rhamnose residues and an unique monosaccharide--anthrose. As anthrose was not found in spores of related strains of bacteria, we envisioned the detection of B. anthracis spores based on antibodies against anthrose-containing polysaccharides. Carbohydrate-protein conjugates containing the synthetic tetrasaccharide, an anthrose-rhamnose disaccharide or anthrose alone were employed to immunize mice. All three formulations were immunogenic and elicited IgG responses with different fine specificities. All sera and monoclonal antibodies derived from tetrasaccharide immunized mice cross-reacted not only with spore lysates of a panel of virulent B. anthracis strains, but also with some of the B. cereus strains tested. CONCLUSIONS: Our results demonstrate that antibodies to synthetic carbohydrates are useful tools for epitope analyses of complex carbohydrate antigens and for the detection of particular target structures in biological specimens. SIGNIFICANCE AND IMPACT OF THE STUDY: Although not strictly specific for B. anthracis spores, antibodies against the tetrasaccharide may have potential as immuno-capturing components for a highly sensitive spore detection system.