Flotation of blocolloids suspended in liquid phases of different compositions and pH values

The flotation capacity was determined for cells of yeasts strains belonging to the genera Hansenula, Candida and Saccharomyces. A heterogeneous group of yeasts, comprising strains from the three genera, was identified as showing high flotation capacities (degrees of flotation above 50%), which were practically not affected by variations in medium pH in both the synthetic medium and 2% molasses. Thus, the flotation capacity of the cells in this yeast group seemed strongly dependent on the liquid phase properties and/or growth medium composition, more than on the simple variation in pH of the cell suspensions. A second group of strains, belonging to the Saccharomyces genus, including also brewing yeast strains, was identified as having lower flotation capacities (degrees of flotation below 50% at pH 1.5), which showed no alterations or variations significantly affected by the medium pH. Foam volumes obtained with Saccharomyces strains were greater in synthetic media than in molasses owing to the higher air flow rates required for flotation in molasses. The flotation efficiency decreased in molasses in all cases as well as the foam volume, except in the case of Hansenula cells, which showed an increased foam volume. This was probably due to variations in product excretion by the different yeasts and/or differences in cell wall composition.

Biochemical characterization of Neurospora crassa glycogenin (GNN), the self-glucosylating initiator of glycogen synthesis

Glycogenin acts in the initiation step of glycogen biosynthesis by catalyzing a self-glucosylation reaction. In a previous work [de Paula et al., Arch. Biochem. Biophys. 435 (2005) 112-124], we described the isolation of the cDNA gnn, which encodes the protein glycogenin (GNN) in Neurospora crassa. This work presents a set of biochemical and functional studies confirming the GNN role in glycogen biosynthesis. Kinetic experiments showed a very low GNN K-m (4.41 mu M) for the substrate UDP-glucose. Recombinant GNN was produced in Escherichia coli and analysis by mass spectroscopy identified a peptide containing an oligosaccharide chain attached to Tyr196 residue. Site-directed mutagenesis and functional complementation of a Saccharomyces cerevisiae mutant strain confirmed the participation of this residue in the GNN self-glucosylation and indicated the Tyr198 residue as an additional, although less active, glucosylation site. The physical interaction between GNN and glycogen synthase (GSN) was analyzed by the two-hybrid assay. While the entire GSN was required for full interaction, the C-terminus in GNN was more important. Furthermore, mutation in the GNN glucosylation sites did not impair the interaction with GSN. (c) 2005 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

Yeast flotation viewed as the result of the interplay of supernatant composition and cell-wall hydrophobicity

Flotation is a process of cell separation based on the affinity of cells to air bubbles. In the present work, flotability and hydrophobicity were determined using cells from different yeasts (Hansenulla polymorpha, Saccharomyces cerevisiae, Candida albicans), which were propagated in different media and at different temperatures. Alterations to the supernatant of the cells were also carried out before the flotation assays. The results described here indicate that supernatants of the yeast cells can play a more important role on flotation than cell-wall hydrophobicity. For example, wall-hydrophobicity of strain FLT-01 of S. cerevisiae was high but flotation did not occur when their washed cells were resuspended in water. Additions of neopeptone to cultures of S. cerevisiae and H. polymorpha repressed flotation and increased the volume of foam. An additional task of the present work was to show that the relationship between cell-wall hydrophobicity and flotation performance was dependent on the method used for the measurement of hydrophobicity. Based on the assay procedure, two types of hydrophobicity were distinguished: (a) the apparent hydrophobicity for cells suspended in the medium and expressed by the degree of cell affinity to the organic solvent in the two-phase system supernatant/hexane; (b) the standard hydrophobicity, which was determined for cells suspended in a standard solution (acetate buffer, in the present work) within the acetate buffer/hexane system. Flotation of cells of S. cerevisiae and C albicans were best related to the degree of apparent hydrophobicity (varying with the supernatant composition at the cell/medium interface) rather than to the degree of standard hydrophobicity (varying with the alterations in the wall components, since the liquid phase was constant in the assay). However, depending on the yeast unpredictable results can be obtained. For example, cells of H. polymorpha exhibited good flotation associated to a high degree of standard hydrophobicity while having a lower degree of apparent hydrophobicity. Concerning growth temperature, flotation of cells of C albicans was strongly repressed when the temperature was raised from 30 to 38 degreesC while a similar effect was not observed in cultures of S. cerevisiae and H. polymorpha. It is difficult to understand and predict flotation of yeast cells but simple modifications made to the supernatant of cultures can activate or repress flotation. (C) 2003 Elsevier B.V. B.V. All rights reserved.

Acetylated DNA-damaging clerodane diterpenes from Casearia sylvestris

In addition to the known diterpene casearin G (1), two new clerodane diterpene casearins type, casearin S (2) and casearin T (3), were isolated from an acetylated bioactive CH2Cl2/MeOH extract from leaves of Casealia sylvestris. The diterpenes 1-3 exhibited moderate but selective activity towards the DNA-repair deficient yeast Saccharomyces cerevisiae mutants RAD 52YK and RS 321. The structures of 1-3 were established on the basis of NMR spectroscopic experiments (C) 1998 Elsevier B.V. Ltd. All rights reserved.

Pectinolytic activity secreted by yeasts isolated from fermented citrus molasses

Aims: the aim of this study was to obtain improved strains of pectinolytic yeasts adapted to the conditions of an industrial fermentation process, which was continuously operated to convert citrus molasses into ethanol.Methods and Results: the starter yeast of the industrial fermentation process was a commercial baker's yeast, which was capable of growing without forming any secretion halo of pectinase activity on solid medium. Nevertheless, isolates showing secretion of pectinolytic activity on plates were obtained from the fermentation process. The secretion of pectin-degrading activity by isolates on plates was repressed by galactose and improved as the result of colony aging on polygalacturonic acid plates at 30 degrees C. Liquefaction of polygalacturonate gels as well as the splitting of the pectin-degrading activity into a wall-linked and a supernatant fraction were also observed when the starter yeast was propagated under agitation in liquid medium containing pectin.Conclusions: Isolates capable of secreting pectinolytic activity on plates were predominant at the end of the citrus molasses fermentation. Nevertheless, the sizes of the secretion haloes on plates were not necessarily an indication of the levels of pectinolytic activity secreted in the liquid medium.Significance and Impact of the Study: Improved pectinolytic strains of Saccharomyces can be used as a source of pectinases for a variety of applications. This organism also participates in plant deterioration processes.

On-line determination of Sb(III) and total Sb using baker's yeast immobilized on polyurethane foam and hydride generation inductively coupled plasma optical emission spectrometry

The yeast Saccharomyces cerevisiae was immobilized in cubes of polyurethane foam and the ability of this immobilized material to separate Sb(III) and Sb(V) was investigated. A method based on sequential determination of total Sb (after on-line reduction of Sb(V) to Sb(III) with thiourea) and Sb(Ill) (after on-line solid-liquid phase extraction) by hydride generation inductively coupled plasma optical emission spectrometry is proposed. A flow system assembled with solenoid valves was used to manage all stages of the process. The effects of pH, sample loading and elution flow rates on solid-liquid phase extraction of Sb(III) were evaluated. Also, the parameters related to online pre-reduction (reaction coil and flow rates) were optimized. Detection limits of 0.8 and 0.15 mu g L-1 were obtained for total Sb and Sb (III), respectively. The proposed method was applied to the analysis of river water and effluent samples. The results obtained for the determination of total Sb were in agreement with expected values, including the river water Standard Reference Material 1640 certified by the National Institute of Standards and Technology (NIST). Recoveries of Sb(III) and Sb(V) in spiked samples were between 81 19 and I I I 15% when 120 s of sample loading were used. (c) 2006 Elsevier B.V. All rights reserved.

Trypanosoma cruzi: identification and characterization of a novel ribosomal protein L27 (TcrL27) that cross-reacts with an affinity-purified anti-Sm antibody

Small nuclear ribonucleoproteins (snRNPs)are involved in trans-splicing processing of pre-mRNA in Trypanosoma cruzi. To clone T. cruzi snRNPs we screened an epimastigote cDNA library with a purified antibody raised against the Sm-binding site of a yeast sequence. A clone was obtained containing a 507 bp-insert with an ORF of 399 bp and coding for a protein of 133 amino acids. Sequence analysis revealed high identity with the L27 ribosomal proteins from different species including: Canis familiaris, Homo sapiens, Schizosaccharomyces pombe and Saccharomyces cerevisiae. This protein has not been previously described in the literature and seems to be a new ribosomal protein in T. cruzi and was given the code TcrL27. To express this recombinant T. cruzi L27 ribosomal protein in E. coli, the insert was subcloned into the pET32a vector and a 26 kDa recombinant protein was purified. Immunoblotting studies demonstrated that this purified recombinant protein was recognized by the same anti-Sm serum used in the library screening as well as by chagasic and systemic lupus erythemathosus (SLE) sera. Our results suggest that the T. cruzi L27 ribosomal protein may be involved in autoimmunity of Chagas disease.

CURRENT ASPECTS OF FUEL ETHANOL-PRODUCTION IN BRAZIL

Brazil has become a great producer of bioethanol using sugarcane as the basic raw material. Fed-batch process and continuous process are used. Biogas generation from vinasse, production of dry yeast, and autolyzed bagasse for animal feed are making the ethanol production less polluting and more profitable. Bagasse surplus has also been converted into electrical energy. Another alternative use for bioethanol is its conversion to petrochemical derivatives. Up to the present, however, this conversion has been carried out on only a small scale by the industry.

Turbinatine, a potential key intermediate in the biosynthesis of corynanthean-type indole alkaloids

Extraction of the leaves of Chimarrhis turbinata has led to the isolation of turbinatine (1), a new corynanthean-type indole alkaloid, besides four known indole alkaloids, strictosidine, 5alpha-carboxystrictosidine, vallesiachotamine, and isovallesiachotamine. The structural determination of 1 was based on 1D and 2D spectroscopic data. An evaluation of the DNA-damaging activities of the isolates was performed by means of a bioassay using mutant strains of Saccharomyces cerevisiae, which indicated these compounds were weakly active.

Altered patterns of maltose and glucose fermentation by brewing and wine yeasts influenced by the complexity of nitrogen source

Maltose and glucose fermentations by industrial brewing and wine yeasts strains were strongly affected by the structural complexity of the nitrogen source. In this study, four Saccharomyces cerevisiae strains, two brewing and two wine yeasts, were grown in a medium containing maltose or glucose supplemented with a nitrogen source varying from a single ammonium salt (ammonium sulfate) to free amino acids (casamino acids) and peptides (peptone). Diauxie was observed at low sugar concentration for brewing and wine strains, independent of nitrogen supplementation, and the type of sugar. At high sugar concentrations altered patterns of sugar fermentation were observed, and biomass accumulation and ethanol production depended on the nature of the nitrogen source and were different for brewing and wine strains. In maltose, high biomass production was observed under peptone and casamino acids for the brewing and wine strains, however efficient maltose utilization and high ethanol production was only observed in the presence of casamino acids for one brewing and one wine strain studied. Conversely, peptone and casamino acids induced higher biomass and ethanol production for the two other brewing and wine strains studied. With glucose, in general, peptone induced higher fermentation performance for all strains, and one brewing and wine strain produced the same amount of ethanol with peptone and casamino acids supplementation. Ammonium salts always induced poor yeast performance. The results described in this paper suggest that the complex nitrogen composition of the cultivation medium may create conditions resembling those responsible for inducing sluggish/stuck fermentation, and indicate that the kind and concentration of sugar, the complexity of nitrogen source and the yeast genetic background influence optimal industrial yeast fermentation performance.

Speciation analysis of Sn(II) and Sn(IV) using baker's yeast and inductively coupled plasma optical emission spectrometry

The use of Saccharomyces cerevisiae as a substrate to selectively retain Sn(II) and Sn(IV) has been investigated. Several factors affecting the retention of the analytes by yeast, such as pH, amount of biomass, temperature and time of contact were evaluated. Based on this study, a method for determination of Sn(II) and Sn(IV) combining inductively coupled plasma optical emission spectrometry (ICP OES) and solid phase extraction using Saccharomyces cerevisiae is proposed. The procedure consists of the selective retention of Sn(IV) by yeast at pH = 2.0 while Sn(II) remains in solution. Determination of tin in the solid phase was easily carried out by submitting a slurry of the yeast (0.5 g/40 mL) directly to ICP OES. The precision of the extraction procedure was characterized by an RSD lower than 4%. The detection limits of tin (3 sigma) in the solid phase and the liquid phase were 1.1 and 0.7 mu g L-1, respectively. The proposed approach was evaluated for determination of Sn(II) and Sn(IV) in spiked river water and real samples of industrial waste water (untreated and treated). For all samples, recoveries of spiked Sn(II) and Sn(IV) were between 85 and 112%.

Determination of Cd(II) and Cd-metallothioneins in biological extracts using baker's yeast and inductively coupled plasma optical emission spectrometry

The use of Saccharomyces cerevisiae as a sorbent material to separate Cd(II) and Cd-metallothionein complex (Cd-MT) has been explored. Solid-liquid phase extractions were carried out in batch mode and the main parameters of the process (pH, temperature, time of incubation, amount of biomass and analyte) were evaluated. Under optimized conditions, the yeast quantitatively retain (94 +/- 5%) the Cd(II) while 97 +/- 2% of the Cd-MT remain in the supernatant. on base of the findings of this study, a simple method is proposed to determine Cd(II) and Cd-MT in cytosols extracted from mouse kidney and crab hepatopancreas. Inductively coupled plasma optical emission spectrometry was used to quantify the analytes in solid and liquid phase. Determination of Cd in the solid phase was carried out by introducing a slurry of the yeast (0.0625 g/10 mL) directly to the inductively coupled plasma optical emission spectrometer. Mixed standards solutions, which also have been submitted to the extraction procedure, were used to quantify the analytes in the samples. Thus, matrix effects due to nebulization of the slurry were overcame. Limits of detection (3 sigma) for Cd(II) and Cd-MT were 1.5 and 1.2 mu g L-1, respectively. Relative standard deviations of signals were 4.2% for measurements in the slurry of solid phase and 2.1% for measurements in the liquid phase. Recoveries of the analytes in cytosol samples were between 76 and 114%. The concentrations of Cd(II) (2.4 +/- 0.5 mu g L-1) and Cd-MT (3.0 +/- 0.5 mu g L-1) found by using the proposed approach were close to those found by tangential-flow ultrafiltration technique (2.6 +/- 0.7 mu g L-1 for Cd(II) and 3.7 +/- 1.7 mu g L-1 for Cd-MT).

Iridoid and seco-iridoid glucosides from Chioccoca alba (Rubiaceae)

Phytochemical investigation of Chioccoca alba afforded three new iridoids, alboside I, alboside II and alboside III, and a new seco-iridoid alboside V. Alboside IV showed moderate activity towards the DNA repair-deficient mutant RS321 of Saccharomyces cerevisiae. The structural elucidation of the new compounds was performed by ES-MS and by 1D and 2D NMR spectroscopic methods. (C) 1999 Elsevier B.V. Ltd. All rights reserved.

NEW YEAST STRAINS FOR ALCOHOLIC FERMENTATION AT HIGHER SUGAR CONCENTRATION

New yeast strains for alcoholic fermentation were isolated from samples collected from Brazilian alcohol factories at the end of the sugar cane crop season. They were selected by their capacity of fermenting concentrated sugar cane syrup as well as high sucrose concentrations in synthetic medium with a conversion efficiency of 89-92%. The strains were identified as Saccharomyces cerevisiae.

Effect of 3,4,4 '-trichlorocarbanilide on growth of lactic acid bacteria contaminants in alcoholic fermentation

3,4,4'-trichlorocarbanilide (TCC) was rested as a new method of bacterial growth control for S. cerevisiae alcoholic fermentations of diluted high test molasses (HTM). Minimal inhibitory concentration (MIC) was tested to determine the necessary concentration of TCC to control bacterial growth. The fed-batch alcoholic fermentation process was used with cell recycle similar to industrial conditions and Lactobacillus fermentum CCT 1407 was mixed in the first inoculum to grow with the yeast. Yeast extract was added into the must to stimulate bacterial growth. The best results of TCC's MIC to bacterial growth of Lactobacillus fermentum and Leuconostoc mesenteroides (< 0.125-1.0 mu g/ml) and Saccharomyces cerevisiae (16 mu g/ml) occurred when it was combined with sodium dodecylsulphate (SDS) in a 1: 4 TCC/SDS ratio (wt/wt) in distilled water solution. 1.8 g/l TCC entrapped in calcium alginate added to the must with yeast extract inhibited the growth of Lactobacillus fermentum CCT 1407 maintaining a controlled acidity, higher yeast viability and up to 20.8% of improvement in the average of alcoholic efficiency. Addition of 0.075 g/l TCC entrapped in calcium alginate and 1.67 mg/l SDS in the wort with yeast extract (0-5.0 g/l), inhibited and controlled the extensive bacterial contamination for 19 cycles of fermentation. (C) 1998 Published by Elsevier B.V. Ltd.