The Influence of Initial Xylose Concentration, Agitation, and Aeration on Ethanol Production by Pichia stipitis from Rice Straw Hemicellulosic Hydrolysate

Rice straw hemicellulosic hydrolysate was used as fermentation medium for ethanol production by Pichia stipitis NRRL Y-7124. Shaking bath experiments were initially performed aiming to establish the best initial xylose concentration to be used in this bioconversion process. In the sequence, assays were carried out under different agitation (100 to 200 rpm) and aeration ((V) under bar (flask)/V(medium) ratio varying from 2.5 to 5.0) conditions, and the influence of these variables on the fermentative parameters values (ethanol yield factor, Y(P/S); cell yield factor, Y(X/S); and ethanol volumetric productivity, Q(P)) was investigated through a 2(2) full-factorial design. Initial xylose concentration of about 50 g/l was the most suitable for the development of this process, since the yeast was able to convert substrate in product with high efficiency. The factorial design assays showed a strong influence of both process variables in all the evaluated responses. The agitation and aeration increase caused a deviation in the yeast metabolism from ethanol to biomass production. The best results (Y(P/S) = 0.37 g/g and Q(P) = 0.39 g/l. h) were found when the lowest aeration (2.5 V(flask)/V(medium) ratio) and highest agitation (200 rpm) levels were employed. Under this condition, a process efficiency of 72.5% was achieved. These results demonstrated that the establishment of adequate conditions of aeration is of great relevance to improve the ethanol production from xylose by Pichia stipitis, using rice straw hemicellulosic hydrolysate as fermentation medium.

Validation of a gas chromatographic method to quantify sesquiterpenes in copaiba oils

Copaifera species (Leguminoseae) are popularly known as ""copaiba"" or ""copaiva"". The oleoresins obtained from the trunk of these species have been extensively used in folk medicine and are commercialized in Brazil as crude oil and in several pharmaceutical and cosmetic products. This work reports a complete validated method for the quantification of beta-caryophyllene, alpha-copaene, and alpha-humulene in distinct copaiba oleoresins available commercially. Thus, essential oil samples (100 mu L) were dissolved in 20 mL of hexanes containing internal standard (1,2,4,5-tetramethylbenzene, 3.0 mM) in a 25 mL glass flask. A 1 mu L aliquot was injected into the GC-FID system. A fused-silica capillary column HP-5, coated with 5% phenylmethylsiloxane was used for this study. The developed method gave a good detection response with linearity in the range of 0.10-18.74 mM. Limits of detection and quantitation variety ranged between 0.003 and 0.091 mM. beta-Caryophyllene, alpha-copaene, and alpha-humulene were recovered in a range from 74.71% to 88.31%, displaying RSD lower than 10% and relative errors between -11.69% and -25.30%. Therefore, this method could be considered as an analytical tool for the quality control of different Copaifera oil samples and its products in both cosmetic and pharmaceutical companies. (C) 2010 Elsevier B.V. All rights reserved.

Ester prodrugs of a potent analgesic, morphine-6-sulfate: syntheses, spectroscopic and physicochemical properties

The aim of this work is to develop 3-acyl prodrugs of the potent analgesic morphine-6-sulfate (M6S). These are expected to have higher potency and/or exhibit longer duration of analgesic action than the parent compound. M6S and the prodrugs were synthesized, then purified either by recrystallization or by semi-preparative HPLC and the structures confirmed by mass spectrometry, IR spectrophotometry and by detailed 1- and 2-D NMR studies. The lipophilicities of the compounds were assessed by a combination of shake-flask, group contribution and HPLC retention methods. The octanol-buffer partition coefficient could only be obtained directly for 3-heptanoylmorphine-6-sulfate, using the shake-flask method. The partition coefficients (P) for the remaining prodrugs were estimated from known methylene group contributions. A good linear relationship between log P and the HPLC log capacity factors was demonstrated. Hydrolysis of the 3-acetyl prodrug, as a representative of the group, was found to occur relatively slowly in buffers (pH range 6.15-8.01), with a small buffer catalysis contribution. The rates of enzymatic hydrolysis of the 3-acyl group in 10% rat blood and in 10% rat brain homogenate were investigated. The prodrugs followed apparent first order hydrolysis kinetics, with a significantly faster hydrolysis rate found in 10% rat brain homogenate than in 10% rat blood for all compounds. (C) 1998 Elsevier Science B.V. All rights reserved.

A phase-segregated model for plant cell culture: The effect of cell volume fraction

Plant cells are characterized by low water content, so the fraction of cell volume (volume fraction) in a vessel is large compared with other cell systems, even if the cell concentrations are the same. Therefore, concentration of plant cells should preferably be expressed by the liquid volume basis rather than by the total vessel volume basis. In this paper, a new model is proposed to analyze behavior of a plant cell culture by dividing the cell suspension into the biotic- and abiotic-phases, Using this model, we analyzed the cell-growth and the alkaloid production by Catharanthus roseus, Large errors in the simulated results were observed if the phase-segregation was not considered.

Involvement of caveolin-2 in caveolar biogenesis in MDCK cells

Caveolins have been identified as key components of caveolae, specialized cholesterol-enriched raft domains visible as small flask-shaped invaginations of the plasma membrane. In polarized MDCK cells caveolin-1 and -2 are found together on basolateral caveolae whereas the apical membrane, where only caveolin-1 is present, lacks caveolae. Expression of a caveolin mutant prevented the formation of the large caveolin-1/-2 hetero-oligomeric complexes, and led to intracellular retention of caveolin-2 and disappearance of caveolae from the basolateral membrane. Correspondingly, in MDCK cells over-expressing caveolin-2 the basolateral membrane exhibited an increased number of caveolae. These results indicate the involvement of caveolin-2 in caveolar biogenesis. (C) 2003 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

Estudo comparativo de procedimentos experimentais e computacionais para cálculo da lipofilia molecular

Introdução – A lipofilia é uma das propriedades físico-químicas que mais influencia a capacidade de uma molécula se movimentar através de compartimentos biológicos. O coeficiente de partição octanol/água (log P) permite, assim, obter uma estimativa da absorção dos fármacos no organismo. A existência de métodos indirectos para um cálculo rápido do log P pode revelar-se de grande importância na análise de listas de compostos com potencial acção farmacológica, reduzindo-as àqueles que se prevêem ter um melhor comportamento biológico. Objectivos – O propósito deste estudo é dar a conhecer um método cromatográfico de RP-HPLC desenvolvido para a determinação indirecta da lipofilia molecular e avaliar a performance de vários programas de cálculo computacional desse mesmo parâmetro. Metodologias – Seleccionaram-se 25 compostos químicos, avaliou-se o log P de cada um deles por RP-HPLC e confrontaram-se os resultados obtidos com os de sete programas computacionais. Resultados – O método RP-HPLC testado demonstrou ser vantajoso em comparação com o convencional shake flask. O programa de cálculo indirecto que proporcionou resultados mais próximos dos experimentais foi o ALOGPS© 2.1. Conclusões – A escolha ideal para a determinação da lipofilia de compostos cujo log P estimado esteja entre 0 e 6 é, sobretudo no que diz respeito à rapidez e simplicidade do processo, o método experimental indirecto RP-HPLC. Quanto aos métodos computacionais concluiu-se que nenhum dos programas, incluindo o ALOGPS© 2.1, demonstrou ser eficaz na avaliação de isómeros pelo que, para estes compostos, será sempre necessário recorrer ao método shake flask ou RP-HPLC.

Production of recombinant protein hLIF in static and dynamic systems of animal cell culture

Thesis for the Degree of Master of Science in Biotechnology Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

Frequency, diversity, and productivity study on the Aedes aegypti most preferred containers in the city of Manaus, Amazonas, Brazil

The most preferred containers by Aedes aegypti were studied April and July (rainy and dry periods) in two Manaus neighbourhoods. In all, 2,700 premises and 13,912 containers were examined, most (87%) recorded outdoors. Out of the 13,100 inspected premises, only 1.6% showed to be positive for Aedes aegypti, summing up to 7,916 collected samples. Most frequently found containers outdoors in either neighbourhood regardless of rain or dry period were Bottles flasks and Storage, and indoors, Fixed, Flowerpots, and buckets. Productivity was estimated according to the number of premises and positive containers investigated, showing the actual container groups productivity. Considering both rainy and dry periods outdoors at Praça 14 the groups of Tyre, Flask, Bottle, Construction Equipment and Fixed, had the highest averages respectively. Construction Equipment and Flask groups were the most productive in Coroado in April. Flask, Construction Equipment and Storage groups stood out in July.

Production of bacterial biopolymers from industrial fat-containing wastes

Dissertation presented in partial fulfilment of the Requirements for the Degree of Master in Biotechnology

Production and characterization of Chitin-Glucan Complex by Komagataella pastoris

This thesis was focused on the production, extraction and characterization of chitin:β-glucan complex (CGC). In this process, glycerol byproduct from the biodiesel industry was used as carbon source. The selected CGC producing yeast was Komagataella pastoris (formerly known as Pichia pastoris), due the fact that to achieved high cell densities using as carbon source glycerol from the biodiesel industry. Firstly, a screening of K. pastoris strains was performed in shake flask assays, in order to select the strain of K. pastoris with better performance, in terms of growth, using glycerol as a carbon source. K. pastoris strain DSM 70877 achieved higher final cell densities (92-97 g/l), using pure glycerol (99%, w/v) and in glycerol from the biodiesel industry (86%, w/v), respectively, compared to DSM 70382 strain (74-82 g/l). Based on these shake flask assays results, the wild type DSM 70877 strain was selected to proceed for cultivation in a 2 l bioreactor, using glycerol byproduct (40 g/l), as sole carbon source. Biomass production by K. pastoris was performed under controlled temperature and pH (30.0 ºC and 5.0, respectively). More than 100 g/l biomass was obtained in less than 48 h. The yield of biomass on a glycerol basis was 0.55 g/g during the batch phase and 0.63 g/g during the fed-batch phase. In order to optimize the downstream process, by increasing extraction and purification efficiency of CGC from K. pastoris biomass, several assays were performed. It was found that extraction with 5 M NaOH at 65 ºC, during 2 hours, associated to neutralization with HCl, followed by successive washing steps with deionised water until conductivity of ≤20μS/cm, increased CGC purity. The obtained copolymer, CGCpure, had a chitin:glucan molar ratio of 25:75 mol% close to commercial CGC samples extracted from A. niger mycelium, kiOsmetine from Kitozyme (30:70 mol%). CGCpure was characterized by solid-state Nuclear Magnetic Resonance (NMR) spectroscopy and Differential Scanning Calorimetry (DCS), revealing a CGC with higher purity than a CGC commercial (kiOsmetine). In order to optimize CGC production, a set of batch cultivation experiments was performed to evaluate the effect of pH (3.5–6.5) and temperature (20–40 ºC) on the specific cell growth rate, CGC production and polymer composition. Statistical tools (response surface methodology and central composite design) were used. The CGC content in the biomass and the volumetric productivity (rp) were not significantly affected within the tested pH and temperature ranges. In contrast, the effect of pH and temperature on the CGC molar ratio was more pronounced. The highest chitin: β-glucan molar ratio (> 14:86) was obtained for the mid-range pH (4.5-5.8) and temperatures (26–33 ºC). The ability of K. pastoris to synthesize CGC with different molar ratios as a function of pH and temperature is a feature that can be exploited to obtain tailored polymer compositions.(...)

Cell functional enviromics: applications to Pichia pastoris

The present PhD thesis develops the cell functional enviromics (CFE) method to investigate the relationship between environment and cellular physiology. CFE may be defined as the envirome-wide cellular function reconstruction through the collection and systems-level analysis of dynamic envirome data. Throughout the thesis, CFE is illustrated by two main applications to cultures of a constitutive P. pastoris X33 strain expressing a scFv antibody fragment. The first application addresses the challenge of culture media development. A dataset was built from 26 shake flask experiments, with variations in trace elements concentrations and basal medium dilution based on the standard BSM+PTM1. Protein yield showed high sensitivity to culture medium variations, while biomass was essentially determined by BSM dilution. High scFv yield was associated with high overall metabolic fluxes through central carbon pathways concomitantly with a relative shift of carbon flux from biosynthetic towards energy-generating pathways. CFE identified three cellular functions (growth, energy generation and by-product formation) that together described 98.8% of the variance in observed fluxes. Analyses of how medium factors relate to identified cellular functions showed iron and manganese at concentrations close to PTM1 inhibit overall metabolic activity. The second application addresses bioreactor operation. Pilot 50 L fed-batch cultivations, followed by 1H-NMR exometabolite profiling, allowed the acquisition of data for 21 environmental factors over time. CFE identified five major metabolic pathway groups that are frequently activated by the environment. The resulting functional enviromics map may serve as template for future optimization of media composition and feeding strategies for Pichia pastoris. The present PhD thesis is a step forward towards establishing the foundations of CFE that is still at its infancy. The methods developed herein are a contribution for changing the culture media and process development paradigm towards a holistic and systematic discipline in the future.

Dynamic wet etching of silicon through isopropanol alcohol evaporation

In this paper, Isopropanol (IPA) availability during the anisotropic etching of silicon in Potassium Hydroxide (KOH) solutions was investigated. Squares of 8 to 40 m were patterned to (100) oriented silicon wafers through DWL (Direct Writing Laser) photolithography. The wet etching process was performed inside an open HDPE (High Density Polyethylene) flask with ultrasonic agitation. IPA volume and evaporation was studied in a dynamic etching process, and subsequent influence on the silicon etching was inspected. For the tested conditions, evaporation rates for water vapor and IPA were determined as approximately 0.0417 mL/min and 0.175 mL/min, respectively. Results demonstrate that IPA availability, and not concentration, plays an important role in the definition of the final structure. Transversal SEM (Scanning Electron Microscopy) analysis demonstrates a correlation between microloading effects (as a consequence of structure spacing) and the angle formed towards the (100) plane.

Avaliação da capacidade de fixação de fósforo pelo solo

Phosphate "fixation" is the convertion of soluble into insoluble phosphate in the soil. There are many factors conditioning phosphate fixation by soil such as reactions originating less soluble compounds (phosphates of iron, aluminum, calcium, magnesium, etc.), PO4-3 adsorption by the colloidal fraction of soils, PO4-3 absorption by the soil microflora, etc. Certain soils of the state of São Paulo (Brazil) are relatively rich in both iron and aluminum oxides. PO4-3 fixation, using P31 and P32 has been verified by researchers, specially with "Terra Roxa". The known methods for fixation evaluation are conventional as this depends on phosphate solution concentration, pH, time of contact between soil and solution, relation of sample weight to solution volume, shaking time, etc. In this experiment, the following conventional method was used: 4 g of soil were shaken for 15 minutes at 30-40 rpm, in 300 ml Erlenmeyer flask in a Wagner shaking machine, together with 100 ml of 0,03 normal phosphate solution (being 0,01 normal as PO4-3 contributed by H8PO4 and 0,02 normal as PO4-3 from KH2PO4). After shaking it was set aside for 24 hours and then filtered. Phosphate was determined in a suitable aliquot of both the original solution (blank) and the soil extract, by the vanadomolibidic-phosphoric acid method. From phosphate concentration in the blank minus phosphate concentration in the soil stract the rate of fixation by 100 g of soil was calculated. The data obtained show that "Terra Roxa" and "Terra Roxa Misturada" have a fairly high PO4-3 fixation capacity, varying from 10 to 24 milliequivalents of PO4-3 per 100 g of soil.

Solubilidade de fosfatos naturais em solução de ácido cítrico a 2%: modificação nas condições de agitação

This paper deals with a modification in the solubilization technique of natural phophates in the 2% citric acid solution. The proposed technique is as follows: 2,5 g of phosphatic material and 250 ml of 2% citric acid solution, in a 500 ml Erlenmeyer flask, are shaken for 30 minutes at 30-40 rpm. The phosphorus (P2O5) was determined by the usual method. The data obtained were compared with the conventional technique in which a Stohmann bottle is used. The natural phosphates used were: Phosphorita de Olinda (Pernambuco), Flórida Phosphate (USA) and Hiperphosphate (África). Statistical analysis was applied to the data and the following conclusions were arrived at: a) The precision is equivalent in both techniques. b) There is no significant variation between the means obtained with the two technique.

Lactacidemia: métodos de dosagem do ácido lático

A critical study of three methods for the determination of lactic acid (EDWARDS, MENDEL & GOLDSCHEIDER, MILLER & MUNTZ) is presented and some modifications are proposed. It was shown t hat more accurate results could be obtained with Edward's technic when an Iena glass filter is connected with the absorption tube. Before the dropping of the permanganate solution it is necessary to pass a current of air through the reaction flask to avoid the oxidation of the non-lactic acid substances which interfere with the reaction. The absorption tube must be maintained at 18°C during the destillation and the titration of the bisulphite binding aldehyde at 4°C. When the sample contains more than 5 mg it is useful to work with greater quantities of the bisulphite. More permanganate is consumed when the lactic acid concentration is higher. The sensivity of the method permits the titration of 0.04 mg to 5 mg of lactic acid in the sample. The calculated error of the method gave 0.018 % and the normal values for blood determined in 20 human cases averaged 10.30 mg per 100 ml (Table VI). MENDEL and GOLDSCHEIDER'S method was modified in the following details: Somogyis deproteinization was performed instead metaphosphoric acid as in the original method; to avoid the evaporation of the acetic aldehyde during the heating time with sulfuric acid a special glass stopped tube is proposed (Fig. 2). The reaction with sulfuric acid and veratrol is performed in an ice bath. Blood proteins precipitants were tried and Somogyi's lattest tecnic showed better results (Table V). Colorimetric readings were done in the PULFRICH photometer using filter S 53 and a 10 mm cup. The method is accurate within an error of 0.23 % and samples of 5 to 70 microg. could be easily determined. Normal values for human blood averaged 10.78 mg per 100 ml. More accurate results were obtained with the technic of MILLER & MUNTZ. Slight modifications were introduced: deproteinization with copper sulfate and sodium tungstate; satured p-hydroxydiphenyl solution according to KOENEMANN which is stable for 5 months when stored in the ice-box. Using the PULFRICH step-photometer the error is 0.17% with samples varying from 0.1 to 10 microg. of lactic acid. The filter employed was S 57 with the 5 mm cup. The method was adapted to 0.1 ml of blood. Normal values for human blood gave an average of 10.58 mg per 100 ml.