Experimental essays on medical and philosophical subjects : particularly, I. On the fermentation of alimentary mixtures, and digestion of the food. II. On the nature and properties of fixed air. III. On the respective powers, and manner of acting, of the different kinds of antiseptics. IV. On the scurvy ; with a proposal for trying new methods to prevent or cure the same, at sea. V. On the dissolvent power of quick-lime ; and a further investigation of the properties of fixed air /

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Comparison of chromic oxide and conventional methods in digestion trials using steers fed pelleted rations /

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Evaluation of lignin contents in tropical forages using different analytical methods and their correlations with degradation of insoluble fiber

We compared the lignin contents of tropical forages by different analytical methods and evaluated their correlations with parameters related to the degradation of neutral detergent fiber (NDF). The lignin content was evaluated by five methods: cellulose solubilization in sulfuric acid [Lignin (sa)], oxidation with potassium permanganate [Lignin (pm)], the Klason lignin method (KL), solubilization in acetyl bromide from acid detergent fiber (ABLadf) and solubilization in acetyl bromide from the cell wall (ABLcw). Samples from ten grasses and ten legumes were used. The lignin content values obtained by gravimetric methods were also corrected for protein contamination, and the corrected values were referred to as Lignin (sa)p, Lignin (pm)p and KLp. The indigestible fraction of NDF (iNDF), the discrete lag (LAG) and the fractional rate of degradation (kd) of NDF were estimated using an in vitro assay. Correcting for protein resulted in reductions (P < 0.05) in the lignin contents as measured by the Lignin (sa), Lignin (pm) and, especially, the KL methods. There was an interaction (P < 0.05) of analytical method and forage group for lignin content. In general, LKp method provided the higher (P < 0.05) lignin contents. The estimates of lignin content obtained by the Lignin (sa)p, Lignin (pm)p and LKp methods were associated (P > 0.05) with all of the NDF degradation parameters. However, the strongest correlation coefficients for all methods evaluated were obtained with Lignin (pm)p and KLp. The lignin content estimated by the ABLcw method did not correlate (P > 0.05) with any parameters of NDF degradation. There was a correlation (P < 0.05) between the lignin content estimated by the ABLadf method and iNDF content. Nonetheless, this correlation was weaker than those found with gravimetric methods. From these results, we concluded that the gravimetric methods produce residues that are contaminated by nitrogenous compounds. Adjustment for these contaminants is suggested, particularly for the KL method, to express lignin content with greater accuracy. The relationships between lignin content measurements and NDF degradation parameters can be better determined using KLp and Lignin (pm)p methods. (C) 2011 Elsevier B.V. All rights reserved.

Improvements to peroxide oxidation methods for analysing sulfur in acid sulfate soils

Improvements to peroxide oxidation methods for analysing acid sulfate soils (ASS) are introduced. The soil solution ratio has been increased to 1 : 40, titrations are performed in suspension, and the duration of the peroxide digest stage is substantially shortened. For 9 acid sulfate soils, the peroxide oxidisable sulfur value obtained using the improved method was compared with the reduced inorganic sulfur result obtained using the chromium reducible sulfur method. Their regression was highly significant, the slope of the regression line was not significantly different (P = 0.05) from unity, and the intercept not significantly different from zero. A complete sulfur budget for the improved method showed there was no loss of sulfur as has been reported for earlier peroxide oxidation techniques. When soils were very finely ground, efficient oxidation of sulfides was achieved, despite the milder digestion conditions. Highly sulfidic and organic soils were shown to be the most difficult to analyse using either the improved method or the chromium method. No single analytical method can be universally applied to all ASS, rather a suite of methods is necessary for a thorough understanding of many ASS. The improved peroxide method, in combination with the chromium method and the 4 M HCl extraction, form a sound platform for informed decision making on the management of acid sulfate soils.

Comparison of different methods for the determination of total organic carbon and humic substances in Brazilian soils

ABSTRACTAiming to compare three different methods for the determination of organic carbon (OC) in the soil and fractions of humic substances, seventeen Brazilian soil samples of different classes and textures were evaluated. Amounts of OC in the soil samples and the humic fractions were measured by the dichromate-oxidation method, with and without external heating in a digestion block at 130 °C for 30 min; by the loss-on-ignition method at 450 °C during 5 h and at 600 °C during 6 h; and by the dry combustion method. Dry combustion was used as reference in order to measure the efficiency of the other methods. Soil OC measured by the dichromate-oxidation method with external heating had the highest efficiency and the best results comparing to the reference method. When external heating was not used, the mean recovery efficiency dropped to 71%. The amount of OC was overestimated by the loss-on-ignition methods. Regression equations obtained between total OC contents of the reference method and those of the other methods showed relatively good adjustment, but all intercepts were different from zero (p < 0.01), which suggests that more accuracy can be obtained using not one single correction factor, but considering also the intercept. The Walkley-Black method underestimated the OC contents of the humic fractions, which was associated with the partial oxidation of the humin fraction. Better results were obtained when external heating was used. For the organic matter fractions, the OC in the humic and fulvic acid fractions can be determined without external heating if the reference method is not available, but the humin fraction requires the external heating.

Multi-elemental analysis of ready-to-eat “baby leaf” vegetables using microwave digestion and high-resolution continuum source atomic absorption spectrometry

The mineral content (phosphorous (P), potassium (K), sodium (Na), calcium (Ca), magnesium (Mg), iron (Fe), manganese (Mn), zinc (Zn) and copper (Cu)) of eight ready-to-eat baby leaf vegetables was determined. The samples were subjected to microwave-assisted digestion and the minerals were quantified by High-Resolution Continuum Source Atomic Absorption Spectrometry (HR-CS-AAS) with flame and electrothermal atomisation. The methods were optimised and validated producing low LOQs, good repeatability and linearity, and recoveries, ranging from 91% to 110% for the minerals analysed. Phosphorous was determined by a standard colorimetric method. The accuracy of the method was checked by analysing a certified reference material; results were in agreement with the quantified value. The samples had a high content of potassium and calcium, but the principal mineral was iron. The mineral content was stable during storage and baby leaf vegetables could represent a good source of minerals in a balanced diet. A linear discriminant analysis was performed to compare the mineral profile obtained and showed, as expected, that the mineral content was similar between samples from the same family. The Linear Discriminant Analysis was able to discriminate different samples based on their mineral profile.

New ultrasonic-based methods in proteomics

Dissertação apresentada para obtenção do Grau de Doutor em Bioquímica pela Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia.A presente dissertação foi preparada no âmbito do convénio bilateral existente entre a Universidade Nova de Lisboa e a Universidade de Vigo.

Analysis of genetic diversity of Trypanosoma cruzi: an application of riboprinting and gradient gel electrophoresis methods

Analysis of restriction fragment length polymorphism (RFLP) profiles derived from digestion of polymerase chain reaction (PCR) products of the ribosomal 18S from Trypanosoma cruzi yields a typical `riboprint' profile that can vary intraspecifically. A selection of 21 stocks of T. cruzi and three outgroup taxa: T. rangeli, T. conorhini and Leishmania braziliensis were analysed by riboprinting to assess divergence within and between taxa. T. rangeli, T. conorhini and L. braziliensis could be easily differentiated from each other and from T. cruzi. Phenetic analysis of PCR-RFLP profiles indicated that, with one or two exceptions, stocks of T. cruzi could be broadly partitioned into two groups that formally corresponded to T. cruzi I and T. cruzi II respectively. To test if ribosomal 18S sequences were homogeneous within each taxon, gradient gel electrophoresis methods were employed utilising either chemical or temperature gradients. Upon interpretation of the melting profiles of riboprints and a section of the 18S independently amplified by PCR, there would appear to be at least two divergent 18S types present within T. cruzi. Heterogeneity within copies of the ribosomal 18S within a single genome has therefore been demonstrated and interestingly, this dimorphic arrangement was also present in the outgroup taxa. Presumably the ancestral duplicative event that led to the divergent 18S types preceded that of speciation within this group. These divergent 18S paralogues may have, or had, different functional pressures or rates of molecular evolution. Whether or not these divergent types are equally transcriptionally active throughout the life cycle, remain to be assessed.

The efficiency of concentration methods used to detect enteric viruses in anaerobically digested sludge

The presence of enteric viruses in biosolids can be underestimated due to the inefficient methods (mainly molecular methods) used to recover the viruses from these matrices. Therefore, the goal of this study was to evaluate the different methods used to recover adenoviruses (AdV), rotavirus species A (RVA), norovirus genogroup II (NoV GII) and the hepatitis A virus (HAV) from biosolid samples at a large urban wastewater treatment plant in Brazil after they had been treated by mesophilic anaerobic digestion. Quantitative polymerase chain reaction (PCR) was used for spiking experiments to compare the detection limits of feasible methods, such as beef extract elution and ultracentrifugation. Tests were performed to detect the inhibition levels and the bacteriophage PP7 was used as an internal control. The results showed that the inhibitors affected the efficiency of the PCR reaction and that beef extract elution is a suitable method for detecting enteric viruses, mainly AdV from biosolid samples. All of the viral groups were detected in the biosolid samples: AdV (90%), RVA, NoV GII (45%) and HAV (18%), indicating the viruses' resistance to the anaerobic treatment process. This is the first study in Brazil to detect the presence of RVA, AdV, NoV GII and HAV in anaerobically digested sludge, highlighting the importance of adequate waste management.

Evaluation of sample preparation methods and optimization of nickel determination in vegetable tissues

Nickel, although essential to plants, may be toxic to plants and animals. It is mainly assimilated by food ingestion. However, information about the average levels of elements (including Ni) in edible vegetables from different regions is still scarce in Brazil. The objectives of this study were to: (a) evaluate and optimize a method for preparation of vegetable tissue samples for Ni determination; (b) optimize the analytical procedures for determination by Flame Atomic Absorption Spectrometry (FAAS) and by Electrothermal Atomic Absorption (ETAAS) in vegetable samples and (c) determine the Ni concentration in vegetables consumed in the cities of Lorena and Taubaté in the Vale do Paraíba, State of São Paulo, Brazil. By means of the analytical technique for determination by ETAAS or FAAS, the results were validated by the test of analyte addition and recovery. The most viable method tested for quantification of this element was HClO4-HNO3 wet digestion. All samples but carrot tissue collected in Lorena contained Ni levels above the permitted by the Brazilian Ministry of Health. The most disturbing results, requiring more detailed studies, were the Ni concentrations measured in carrot samples from Taubaté, where levels were five times higher than permitted by Brazilian regulations.

Addressing trypsin bias in large scale (phospho)proteome analysis by size exclusion chromatography and secondary digestion of large post-trypsin peptides.

In the vast majority of bottom-up proteomics studies, protein digestion is performed using only mammalian trypsin. Although it is clearly the best enzyme available, the sole use of trypsin rarely leads to complete sequence coverage, even for abundant proteins. It is commonly assumed that this is because many tryptic peptides are either too short or too long to be identified by RPLC-MS/MS. We show through in silico analysis that 20-30% of the total sequence of three proteomes (Schizosaccharomyces pombe, Saccharomyces cerevisiae, and Homo sapiens) is expected to be covered by Large post-Trypsin Peptides (LpTPs) with M(r) above 3000 Da. We then established size exclusion chromatography to fractionate complex yeast tryptic digests into pools of peptides based on size. We found that secondary digestion of LpTPs followed by LC-MS/MS analysis leads to a significant increase in identified proteins and a 32-50% relative increase in average sequence coverage compared to trypsin digestion alone. Application of the developed strategy to analyze the phosphoproteomes of S. pombe and of a human cell line identified a significant fraction of novel phosphosites. Overall our data indicate that specific targeting of LpTPs can complement standard bottom-up workflows to reveal a largely neglected portion of the proteome.

A new method for the determination of plutonium and americium using high pressure microwave digestion and alpha-spectrometry or ICP-SMS

Plutonium and americium are radionuclides particularly difficult to measure in environmental samples because they are alpha-emitters and therefore necessitate a careful separation before any measurement, either using radiometric methods or ICP-SMS. Recent developments in extraction chromatography resins such as Eichrom (R) TRU and TEVA have resolved many of the analytical problems but drawbacks such as low recovery and spectral interferences still occasionally occur. Here, we report on the use of the new Eichrom (R) DGA resin in association with TEVA resin and high pressure microwave acid leaching for the sequential determination of plutonium and americium in environmental samples. The method results in average recoveries of 83 +/- 15% for plutonium and 73 +/- 22% for americium (n = 60), and a less than 10% deviation from reference values of four IAEA reference materials and three samples from intercomparisons exercises. The method is also suitable for measuring Pu-239 in water samples at the mu Bq/l level, if ICP-SMS is used for the measurement.

Accelerated digestion for high-throughput proteomics analysis of whole bacterial proteomes.

In bottom-up proteomics, rapid and efficient protein digestion is crucial for data reliability. However, sample preparation remains one of the rate-limiting steps in proteomics workflows. In this study, we compared the conventional trypsin digestion procedure with two accelerated digestion protocols based on shorter reaction times and microwave-assisted digestion for the preparation of membrane-enriched protein fractions of the human pathogenic bacterium Staphylococcus aureus. Produced peptides were analyzed by Shotgun IPG-IEF, a methodology relying on separation of peptides by IPG-IEF before the conventional LC-MS/MS steps of shotgun proteomics. Data obtained on two LC-MS/MS platforms showed that accelerated digestion protocols, especially the one relying on microwave irradiation, enhanced the cleavage specificity of trypsin and thus improved the digestion efficiency especially for hydrophobic and membrane proteins. The combination of high-throughput proteomics with accelerated and efficient sample preparation should enhance the practicability of proteomics by reducing the time from sample collection to obtaining the results.

The Methods of Possible Joint Treatment of Manure and Sewage Sludge in the Leningrad Region

The nutrient load to the Gulf of Finland has started to increase as a result of the strong economic recovery in agriculture and livestock farming in the Leningrad region. Also sludge produced from municipal wastewater treatment plant of the Leningrad region causes the great impact on the environment, but still the main options for its treatment is disposal on the sludge beds or Landfills. The aim of this study was to evaluate the implementation of possible joint treatment methods of manure form livestock and poultry enterprises and sewage sludge produced from municipal wastewater treatment plants in the Leningrad region. The study is based on published data. The most attention was put on the anaerobic digestion and incineration methods. The manure and sewage sludge generation for the whole Leningrad region and energy potential produced from their treatment were estimated. The calculations showed that total amount of sewage sludge generation is 1 348 000 t/a calculated on wet matter and manure generation is 3 445 000 t/a calculated on wet matter. The potential heat release from anaerobic digestion process and incineration process is 4 880 000 GJ/a and 5 950 000 GJ/a, respectively. Furthermore, the work gives the overview of the general Russian and Finnish legislation concerning manure and sewage sludge treatment. In the Gatchina district it was chosen the WWTP and livestock and poultry enterprises for evaluation of the centralized treatment plant implementation based on anaerobic digestion and incineration methods. The electricity and heat power of plant based on biogas combustion process is 4.3 MW and 7.8 MW, respectively. The electricity and heat power of plant based on manure and sewage sludge incineration process is 3.0 MW and 6.1 MW, respectively.

Développement de méthodes analytiques de séparation des produits de digestion enzymatique des dérivés de cellulose

La cellulose et ses dérivés sont utilisés dans un vaste nombre d’applications incluant le domaine pharmaceutique pour la fabrication de médicaments en tant qu’excipient. Différents dérivés cellulosiques tels que le carboxyméthylcellulose (CMC) et l’hydroxyéthylcellulose (HEC) sont disponibles sur le commerce. Le degré de polymérisation et de modification diffèrent énormément d’un fournisseur à l’autre tout dépendamment de l’origine de la cellulose et de leur procédé de dérivation, leur conférant ainsi différentes propriétés physico-chimiques qui leurs sont propres, telles que la viscosité et la solubilité. Notre intérêt est de développer une méthode analytique permettant de distinguer la différence entre deux sources d’un produit CMC ou HEC. L’objectif spécifique de cette étude de maitrise était l’obtention d’un profil cartographique de ces biopolymères complexes et ce, par le développement d’une méthode de digestion enzymatique donnant les oligosaccharides de plus petites tailles et par la séparation de ces oligosaccharides par les méthodes chromatographiques simples. La digestion fut étudiée avec différents paramètres, tel que le milieu de l’hydrolyse, le pH, la température, le temps de digestion et le ratio substrat/enzyme. Une cellulase de Trichoderma reesei ATCC 26921 fut utilisée pour la digestion partielle de nos échantillons de cellulose. Les oligosaccharides ne possédant pas de groupements chromophores ou fluorophores, ils ne peuvent donc être détectés ni par absorbance UV-Vis, ni par fluorescence. Il a donc été question d’élaborer une méthode de marquage des oligosaccharides avec différents agents, tels que l’acide 8-aminopyrène-1,3,6-trisulfonique (APTS), le 3-acétylamino-6-aminoacridine (AA-Ac) et la phénylhydrazine (PHN). Enfin, l’utilisation de l’électrophorèse capillaire et la chromatographie liquide à haute performance a permis la séparation des produits de digestion enzymatique des dérivés de cellulose. Pour chacune de ces méthodes analytiques, plusieurs paramètres de séparation ont été étudiés.