The methylotrophic yeast Pichia pastoris has become an efficient expression system for heterologous protein production. Different methods have been studied to enhance cell growth as well as the production of products of interest. Two of the major strategies for improving the product or biomass yields are optimizing bioprocess controls and cultivation conditions. In this work, the characteristics of this yeast system and of its different promoters are discussed, and the effect of operational strategies on cell growth and recombinant protein expression is also studied. The effect of different feeding strategies were studied and optimized for pGAP (glyceraldehyde-3-phosphate dehydrogenase)-regulated phytase production in P. pastoris. Alternative carbon sources were screened and the feasibility of using citric acid as a carbon source for recombinant protein production was also investigated. The effects of parameters such as the carbon source concentration and culture pH were studied using shake-flasks, and the effect of different feeding profiles on bioreactor performance was also investigated. Three feeding strategies, Stepwise feeding, Exponential feeding and DO-stat feeding were tested and DO-stat was found to be more efficient and led to a high phytase activity. A modified DO-stat method was investigated to overcome the oxygen limited condition in the standard DO-stat method. For the carbon source, citric acid showed promise in improving phytase expression. Further experiments in bioreactors performed with the presence of certain amount of citric acid showed that less glycerol could be used to achieve the same level of phytase activity.

>Magister Scientiae - MSc / Unavailability and contamination of available water resources are major factors contributing to adverse health conditions worldwide. AgNPs present a potential strategy for water purification; however, their ability to accumulate in organs such as the kidneys, lungs and spleen is a possible source of toxicity. This study investigates the toxicity of AgNPs to Saccharomyces cerevisiae (S. cerevisiae). S. cerevisiae is an excellent model organism for assessing toxic compounds that affect eukaryotic organisms due to their ease of cultivation. AgNPs were prepared by photo-reduction of silver nitrate with OSRAM Vitalux lamp (300 W and 230 V) in the presence of stabilizing agents such as polyvinylpyrrolidone and citric acid, yielding AgNPs. The effects of varying the concentration of the stabilizing agent, time of exposure to the light source, and pH were investigated. The formation of AgNPs was analysed by ultra-violet spectroscopy (UV-Vis) and transmission electron microscope techniques. The results showed that the AgNPs absorbed ultra-violet radiation between 400 and 500 nm and TEM images showed the particles to be both spherical and needle-like in shape. The shapes of the AgNPs were largely dependent on the synthesis method applied. The toxicity of AgNPs was assessed using metabolic activity of yeast cells as biomarker andmonitored with of the chromogenic assay, XTT. S. cerevisiae was introduced into different concentrations of AgNPs and incubated at 37oC for 72 h. After the incubation, XTT assay was performed to assess the cell viability. The XTT results showed that high concentration of AgNPs (100 µg/mL) inhibited the growth of S. cerevisiae. The synthesis of AgNPs and theassessment of their toxicity on S. cerevisiae was thus undertaken and established in this work.

Um das Verhalten von Actiniden im Menschen (Stoffwechsel), in geologischen und in biologischen Systemen vorherzusagen, ist es erforderlich deren Speziation genau zu kennen. Zur Bestimmung dieser wird das chemische Verhalten des Urans hinsichtlich Komplexbildungsreaktionen und Redoxreaktionen in Modellsystemen untersucht. Anhand der gewonnenen thermodynamischen Konstanten und dem Redoxverhalten können Risikoabschätzungen für das jeweilige untersuchte System getroffen werden. Das umweltrelevante Uran(IV)-Uran(VI)-Redoxsystem besitzt mit der metastabilen fünfwertigen Oxidationsstufe einen zumeist kurzlebigen Zwischenzustand. Innerhalb dieser Arbeit gelang es erstmalig die Uran(V)-Fluoreszenz mittels laserspektroskopischer Methoden nach zu weisen. Beispielsweise konnte das Bandenmaximum von aquatischem Uranyl(V) im perchlorhaltigem Medium (λex = 255 nm) mit 440 nm, bei einer Fluoreszenzlebensdauer von 1,10 ± 0,02 μs bestimmt werden. Die fluoreszenzspektroskopische Untersuchung eines aquatischen [U(V)O2(CO3)3]5--Komplexes (λex= 255 nm und 408 nm) zeigte bei Raumtemperatur keine Fluoreszenz. Durch Anwendung der Tieftemperaturtechnik wurden bekannte Quencheffekte des Carbonats unterdrückt, so dass bei beiden Anregungswellenlängen ein für Uran(V) typisches Fluoreszenzspektrum im Bereich von 375 nm bis 450 nm, mit Bandenmaxima bei 401,5 nm (λex = 255 nm) und 413,0 nm (λex = 408 nm) detektiert werden konnte. Darüber hinaus konnte bei 153 K (λex = 255 nm) eine Fluoreszenzlebensdauer von 120 ± 0,1 μs bestimmt werden. Untersetzt wurden diese fluoreszenzspektroskopischen Nachweise durch mikroskopische Studien verschiedener Uran(IV)-Festphasen (Uraninit…UO2, Uran(IV)-Tetrachlorid…UCl4) und einer sulfathaltigen Uran(IV)-Lösung (UIVSO4). Diese wurden durch kontinuierliche Sauerstoffzufuhr zu Uran(VI) oxidiert. Die ablaufende Oxidation wurde mit dem konfokalen Laser Scanning Mikroskop (CLSM) verfolgt, wobei die Proben mit einer Wellenlänge von 408 nm zur Fluoreszenz angeregt wurden. Die auftretenden Bandenmaxima bei 445,5 nm (UO2), bei 445,5 nm (UCl4) und bei 440,0 nm (UIVSO4) konnten eindeutig der Uran(V)-Fluoreszenz zugeordnet werden. Zur Bestimmung thermodynamischer Konstanten mit Hilfe der Tieftemperaturfluoreszenz wurde zunächst der Einfluss der Temperatur auf das Fluoreszenzverhalten des freien Uranyl(VI)-Ions näher betrachtet. Es zeigte sich, dass mit Erwärmung der Probe (T>298 K) die Fluoreszenzlebensdauer von 1,88 μs (298 K) deutlich absinkt. Die Fluoreszenzintensität verringerte sich dabei um 2,3 % pro 1 K zwischen 273 K und 313 K. Im Gegensatz dazu, steigt die Fluoreszenzlebensdauer um das 150-fache auf 257,9 μs bei einer Verminderung der Temperatur (T <298 K) auf 153 K. Das weitere Absenken der Temperatur (T <153 K) zeigte keinen Einfluss auf die Fluoreszenzlebensdauer. Die Lage der Hauptemissionsbanden des freien Uranyl(VI)-Ions (488,0 nm, 509,4 nm, 532,4 nm, 558,0 nm, 586,0 nm) zeigte bei diesen Untersuchungen keine temperaturabhängige Verschiebung. Die Validierung der Tieftemperaturtechnik zur Bestimmung thermodynamischer Konstanten mittels zeitaufgelöster laserinduzierten Fluoreszenzspektroskopie erfolgte anhand des Uran(VI)-Citrat-Systems. Im Gegensatz zu bisherigen fluoreszenzspektroskopischen Betrachtungen bei Raumtemperatur wurde das Fluoreszenzsignal bei tiefen Temperaturen mit einsetzender Komplexierung nicht gequencht, woraus die Ausprägung einer gut interpretierbaren Fluoreszenz resultierte. Die Analyse der spektralen Daten mit SPECFIT ergaben mit log β 101 = 7,24 ± 0,16 für den [UO2(Cit)]--Komplex und log β 202 = 18,90 ± 0,26 für den [(UO2)2(Cit)2]2--Komplex exakt die in der Literatur angegebenen Stabilitätskonstanten. Zudem konnten Einzelkomponentenspektren mit Bandenmaxima bei 475,3 nm, 591,8 nm, 513,5 nm, 537,0 nm und 561,9 nm für den 1:0:1-Komplex und 483,6 nm, 502,7 nm, 524,5 nm, 548,1 nm und 574,0 nm für den 2:0:2-Komplex und Fluoreszenzlebensdauern von 79 ± 15 μs (1:0:1) und 10 ± 3 μs (2:0:2) bestimmt werden. Zur Modellkomplexierung des Uran-Citrat-Systems wurde in dieser Arbeit auch das Komplexbildungsverhalten von U(IV) in Gegenwart von Citronensäure untersucht. Hierbei wurden über den gesamten pH-Wertbereich gelöste Uran-Citrat-Spezies spektroskopisch nachgewiesen und die Stabilitätskonstanten sowie die Einzelkomponentenspektren für die neu gebildeten Uran(IV) und (VI)-Spezies bestimmt. Für die neu gebildeten Citrat-Komplexe des sechswertigen Urans wurden Komplexbildungskonstanten von log β 203 = 22,67 ± 0,34 ([(UO2)2(Cit)3]5-) und log β 103 = 12,35 ± 0,22 ([UO2(Cit)3]7-) und für die Komplexe des vierwertigen Urans von log β 1-21 = -9,74 ± 0,23 ([U(OH)2Cit]-) und log β 1-31 = -20,36 ± 0,22 ([U(OH)3Cit]2-) bestimmt. Untersuchungen zum Redoxverhalten von Uran in Gegenwart von Citronensäure zeigten unter aeroben und anaeroben Versuchsbedingungen eine photochemische Reduktion vom U(VI) zu U(IV), welche spektroskopisch nachgewiesen werden konnte. Dabei zeigt speziell die Reaktion unter oxidierenden Bedingungen, welchen großen Einfluss vor allem organischen Liganden auf das chemische Verhalten des Urans haben können. Sowohl die Reduktion unter O2- als auch die unter N2-Atmosphäre, weisen ein Maximum bei einem pH-Wert von 3,5 bis 4 auf. Unter anaeroben Bedingungen reduziert die Citronensäure mit ca. 66 %, 14 % mehr Uran(VI) zu Uran(IV) als unter anaeroben Bedingungen mit ca. 52 %. Ab einem pH-Wert von 7 konnte eine Reduktion nur unter sauerstofffreien Bedingungen festgestellt werden. Die Wechselwirkung von U(VI) in Gegenwart von Glucose wurde hinsichtlich Reduktion und Komplexierung des Uran(VI) betrachtet. Mit Hilfe der zeitaufgelösten laserinduzierten Fluoreszenzspektroskopie bei tiefen Temperaturen wurde dabei ein Uranyl(VI)-Glucose-Komplex nachgewiesen. Die Komplexierung wurde lediglich bei pH 5 beobachtet und weist eine Komplexbildungskonstante von log β I=0,1 M = 15,25 ± 0,96 für den [UO2(C6H12O6)]2+-Komplex auf. Mit einer Fluoreszenzlebensdauer von 20,9 ± 2,9 μs und den Hauptemissionsbanden bei 499,0nm, 512,1 nm, 525,2 nm, 541,7 nm und 559,3 nm konnte der Uranyl(VI)-Glucose-Komplex fluoreszenzspektroskopisch charakterisiert werden. Unter reduzierenden Bedingungen wurde, ab pH-Wert 4 eine auftretende Umwandlung vom sechswertigen zum vierwertigen Uran durch Glucose in Gegenwart von Licht beobachtet. Der Anteil an gebildetem Uran(IV) steigt asymptotischen bis zu einem pH-Wert von 9, wo das Maximum mit 16 % bestimmt wurde. Als Reaktionsprodukt der Redoxreaktion wurde eine Uran(VI)-Uran(IV)-Mischphase mit der Summenformel [UIV(UVIO2)5(OH)2]12+ identifiziert. Mit Hilfe der cryo-TRLFS wurde, durch Verminderung von Quencheffekten die Uranspeziation in natürlichen Medien (Urin, Mineralwasser) direkt bestimmt. Proben mit Uran-Konzentrationen von < 0,1 μg/L konnten dadurch analysiert werden. In handelsüblichen Mineralwässern wurde die zu erwartende Komplexierung durch Carbonat nachgewiesen. Im Urin zeigte sich in Abhängigkeit vom pH-Wert eine unterschiedliche Uranspeziation. Die fluoreszenzspektroskopische Untersuchung wies bei niedrigerem pH-Wert (pH<6) eine Mischung aus Citrat- und Phosphat-Komplexierung des U(VI) und bei höheren pH-Wert (pH>6) eine deutliche Beteilung von Carbonat an der Komplexierung auf. Diese Ergebnisse stehen in sehr guter Übereinstimmung mit theoretischen Modellrechnungen zur Uranspeziation im Urin. Die in dieser Arbeit gewonnenen Ergebnisse zeigen, dass für eine zuverlässigere Prognose des Urantransportes in Geo- und Biosphäre in Zukunft nicht nur Betrachtungen zur Komplexchemie, sondern auch zum Redoxverhalten des Urans nötig sind, um die Mobilität in der Natur richtig abschätzen zu können.

This research enthusiastically highlights the bio-adsorption of methylene blue (MB) by local, poultry, NaOH and citric acid modified ubiquitous eggshell (LES, NLES, CLES, PES, NPES and CPES) adsorbents. The microstructures of these adsorbents indicated that they had some surface functional moieties that were responsible for the adsorption of MB. The Langmuir isotherm and PSO model best fit the experiment data. The largest Langmuir monolayer adsorption capacity qmax, was 242.47mg/g, with the largest MB initial concentration of 400mg/L. This was a clear indication and a confirmation that MB adsorption by the powdered eggshells was chemisorptive. Moreover, the values of F $F$, the thickness of the boundary layer/film were >0, showing that the rate limiting step for the adsorption process was controlled by more than one diffusion mechanism. The values of ΔG° for the adsorption of MB by the adsorbents indicated that the adsorption reactions were all non-feasible and non-spontaneous. The values for ΔS° (J/K/mol) for LES, NLES and CPES for the uptake of MB showed decrease in the chaos or degree of randomness of the adsorption reactions, and the reverse was the case for PES, NPES and CLES for the uptake of MB, which showed increase in the chaos or degree of randomness of the adsorption. The adsorption of MB by LES, NLES and CPES gave ΔH°(kJ/mol) values which were indicative of endothermic nature of the adsorption systems, and the reverse was the case for the uptake of MB by PES, NPES and CLES, which was indicative of the exothermic nature of the adsorption systems.

This research was undertaken for the purpose of developing a process for producing citric and gluconic acids from plantation molasses. There is an evident need for such a process. At the present time, these acids are being produced by processes in which refined sugar is the principal raw material. If molasses could be used in place of refined sugar, the cost of raw materials would be reduced by approximately ninety percent. In addition, a waste product would be utilized. The scope of this problem is very broad. Its solution will involve the solving of many problems of widely varying natures. In this work it is the aim of the writer to survey the entire field, touching lightly on each of its various phases, rather than to attempt to work out the complete solution of any particular phase of the problem. Since this is the initial research such a course of action seems prudent. It would be useless to solve one phase of the problem without making certain that some other one does not present a serious barrier to the process as a whole. Also, it is only by doing this survey work that the relative importance of the various problems can be determined. In order that he may be acquainted with the economic aspects of the problem, the reader should have some knowledge of the uses of the acids to be produced. The uses of citric acid are well known and need not be discussed here. Gluconic acid, however, has only recently become of any commercial importance. A brief discussion of some of its possible uses will be in order. Many of the uses of gluconic acid are based on its ability to form inner anhydrides, known as lactones, which will regenerate gluconic acid when dissolved in water. The rate of formation of the acid from the lactone may be controlled within certain limits by varying the conditions of temperature and concentration. This property makes it desirable to use gluconic lactone in fruit powders for jelly making, in baking powders, in the manufacture of cell concrete and insulating brick, and, in short, wherever the slow and controllable formation of an acid is wanted. Gluconates are used in the preparation of homogeneous pastes such as dentifrices. Calcium and magnesium gluconates are quite satisfactory polishing agents. The gluconate is the most satisfactory calcium salt for use in medicine, being assimilable, practically tasteless, and non-irritating to tissues. It may be administered by the mouth or by subcutaneous injection. A colloidal suspension of hydrated aluminum oxide in gluconic acid finds use in tanning, furnishing a white, flexible, and durable leather, which is not leached out or stiffened by prolonged treatment with hot water.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第19776号 / 農博第2172号 / 新制||農||1041(附属図書館) / 学位論文||H28||N4992(農学部図書室) / 32812 / 京都大学大学院農学研究科森林科学専攻 / (主査)教授 金山 公三, 教授 矢野 浩之, 教授 五十田 博 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第20766号 / 農博第2249号 / 新制||農||1054(附属図書館) / 学位論文||H29||N5086(農学部図書室) / 京都大学大学院農学研究科森林科学専攻 / (主査)教授 金山 公三, 教授 矢野 浩之, 教授 吉村 剛 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

No / The dissociation constant is one of the most important characteristics of a pharmaceutical chemical moiety which has to be estimated with accuracy. The development of in-situ speciation methods in solutions with parallel measurements using Raman spectroscopy (molecular) and pH (macroscopic) for the identification, characterization, and quantitative determination of citric acid species in aqueous solution by numerical data treatment using a multiwavelength curve fitting program over a range of pH values is described. As a result, the first, second and third stepwise dissociation constants of citric acid have been evaluated as 3.02±0.06, 4.78±0.06 and 6.02±0.04, respectively. From these data over the pH range 2.38-6.16 an excellent agreement with literature values was achieved.

Five experiments were conducted, utilizing 512 crossbred weanling pigs to determine the P (Exp. 1) and Ca (Exp. 2 and 3) equivalency values of microbial phytase based on performance, rib mineralization and P and Ca digestibility estimates, and to investigate the possible interactions of phytase and citric acid (Exp. 4 and 5). In Exp. 1, adding phytase to low P diets linearly increased ADG (P < .001), rib shear force (P < .01), shear energy (P < .02), ash weight (P < .001) and ash percent (P < .001), Ca (P < .001) and P (P < .001) digestibility and digestible Ca (P < .001) and P (P <.001). Added P linearly increased ADG (P < .003), rib shear force (P < .003) shear energy (P < .001), ash weight (P < .001) and ash percent (P < .01), Ca (P < .02) and P (P < .001) digestibility and digestible Ca (P < .02) and P (P <.001). Based on phytase and P linear or nonlinear response equations for ADG, rib shear force, shear energy, and ash weight, P digestibility, and digestible P, the average equivalency of 500 U/kg of phytase was .78 g of P per kg of diet. In Exp. 2, dietary addition of phytase linearly increased rib ash % (P < .03), Ca (P < .001) and P (P < .001) digestibilities, and digested Ca (P < .001) and P (P < .001), but had no effect (P > .10) on ADG and rib shear force and ash weight. Added Ca linearly increased ADG (wk 3-4, P < .04), and rib shear force (P < .001), ash percentage (P < .001) and ash weight (P < .01), and digested Ca (P < .001), but P digestibility (P = .07) and digested P (P = .08) were numerically decreased. In Exp. 3, added phytase linearly increased ADG (wk 3-4, P < .002), feed efficiency (wk 3-4, P < .02), rib ash weight (P < .001), Ca total tract digestibility (P < .001), and Ca (P < .001) and P (P < .001) ileal digestibilities. Added Ca linearly increased ADG (wk 3-4, P < .02), feed efficiency (wk 3-4, P < .01), rib ash percentage (P < .001) and ash weight ( P < .001), shear force (P < .03) and energy (P < .008), and total tract (P < .001) and ileal (P < .001) digestible Ca. Based on phytase and Ca linear or nonlinear response equations for ADG in wk 3-4, measurements of rib mineralization, and digestible Ca, 500 U of microbial phytase was estimated to be equivalent to 1.08 g and .78 g of Ca in Exp. 2 and 3, respectively. In Exp. 4 and 5, dietary phytase addition linearly increased rib shear force (P < .004 and P < .02), shear energy (P < .001), dry bone weight (P < .001), ash weight (P < .001) and ash percent (P < .001). Calcium (P < .001) and P (P < .001) digestibilities were also improved in both experiments when phytase was added. Addition of citric acid in both experiments, reduced dietary pH and stomach digesta pH (P < .05). The addition of citric acid improved ADG (P < .05), feed efficiency (P < .04) and Ca digestibility (P < .05) in Exp. 4, but decreased Ca digestibility in Exp. 5 and had no effect on performance. In Exp. 5, the addition of 2.0% citric acid to the diet supplemented with 500 U/kg of phytase caused a decrease (P < .04) in the phytase activity recovered in the stomach digesta resulting in a phytase by citric acid interaction (P < .02). In summary, the addition of 500 U/kg microbial phytase to weanling pig diets, causes the release of approximately .78 g of P and .93 g of Ca, thus decreasing the need for supplemental P and Ca. The addition of citric acid to phytase supplemented diets does not appear to enhance the efficacy of microbial phytase based on the results of these studies. / Master of Science

Thesis (M.Sc. Agriculture (Horticulture)) -- University of Limpopo, 2022 / The most common postharvest limitation of litchi fruit is pericarp browning, which leads to consumer rejection of the solid fruit in the market even when the edible portion is not affected. Previously, sulphur dioxide (SO2) fumigation was used to control the browning and extend shelf life of litchi fruit. However, SO2 fumigation leaves undesirable residues, alters the fruit taste and may results in health hazards for consumers. An alternative method, namely, minimal processing was used to control pericarp browning and curb postharvest losses in litchi. Litchi fruit were harvested at two maturity stages (early harvest; 120 days after full bloom (DAFB), late harvest; 130 DAFB), peeled and immersed for two (2) minutes in three (3) solutions that represented treatments, namely 1) 1% citric acid 2) 1% calcium lactate and 3) a combination of citric acid and calcium lactate both at 1% measure. The untreated arils were dipped in sodium hypochloride (NaOCl) solution for 1 minute and represented the control samples. The treated arils were packed in sterilized clamshell containers and stored at 1±0.5°C and 95% relative humidity for 12 days, then held at 10±0.5°C for 2 days for shelf life study. As a result of the interaction effect of harvest stages and postharvest pre-treatments, least mass loss percentage (1.32%), juice leakage (1.8 ml per 120 g of fruit) and pH (4.18) was observed in litchi arils harvested late and treated with 1% citric acid only under cold storage. Under shelf life study, H2 control samples presented lower mass loss (2.8%) and juice leakage (4.2 ml per 120 g of fruit). At the end of cold storage, litchi arils harvested early and treated with 1% citric acid combined with 1% calcium lactate presented better tissue strength (56.0 N) and radical scavenging activity (36.6 mmol AAE/mL), while those harvested late presented higher ascorbic acid content (72.9 µg/mL), least microbial population and total colour change (3.5). However, at the end of shelf life storage, litchi arils harvested early and treated with 1% citric acid combined with 1% calcium lactate presented lower (3.1) total change in colour. Overall, harvesting the fruit late and treating with citric acid alone or combined with calcium lactate showed the potential of maintaining better aril quality with least microbial population for up to 12 days under 1±0.5°C storage, whereas harvesteing the fruit early and treating with citric acid alone or combined with calcium lactate showed the potential of maintaining better aril quality under shelf life storage. Keywords: Litchi chinesis sonn; calcium lactate; citric acid; maturity; fresh-cut. / Agricultural Research Council (ARC), National Research Foundation (NRF) and Agri-Seta