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Quantitative estimation of diphtheria and tetanus toxoids. 2. Single radial immuno-diffusion tests (Mancini) and rocket immuno-electrophoresis test in comparison with the flocculation test.

The concentration in Lf units, of an unknown diphtheria or tetanus toxoid preparation is estimated in the flocculation test relative to reference preparations of tetanus and diphtheria antitoxins, respectively. By replacing the antitoxin reference preparations with toxoid reference preparations it should be possible to use immunological methods other than the flocculation test for the quantitative estimation of toxoids in Lf units. A number of diphtheria and tetanus toxoids were tested by rocket immuno-electrophoresis and single radial immuno-diffusion (Mancini test). The concentrations of the unknown toxoids were expressed relative to a diphtheria toxoid calibrated in Lf units (DIFT) and a tetanus toxoid calibrated in Lf units (TEFT), respectively. These two toxoid preparations are regarded as candidates for establishment as international standard preparations. The results obtained in the two tests were compared with those obtained in the flocculation test. In most cases the differences between the results did not exceed 10%. It is concluded, therefore, that the rocket electrophoresis or the radial immuno-diffusion tests can be used as alternatives to the flocculation test.

Diphtheria Toxoid↗

[Isolation and characterization of a flocculating mutant of Saccharomyces diastaticus].

A flocculating strain of Saccharomyces diastaticus NRRL2416 was isolated after ethidium bromide mutagenesis and density gradient separation. Flocculation did not change the general characteristics of the strain. Flocculation was better on maltodextrin-containing media than on others. It was found to be calcium-dependent and sensitive to growth conditions. The deflocculating effects of some sugars suggested that mannose may be involved in the mechanism of flocculation, as for strains of the FLO1 phenotypic group.

Culture Media↗

Effect of mineral nutrients on cell growth and self-flocculation of Tolypothrix tenuis for the production of a biofertilizer.

The influence of mineral nutrients on the growth and self-flocculation of Tolypothrix tenuis was studied. The identification of possible limiting nutrients in the culture medium was performed by the biomass elemental composition approach. A factorial experimental design was used in order to estimate the contribution of macronutrients and micronutrients, as well as their interactions. Iron was identified to be limiting in the culture medium. The micronutrients influenced mainly cellular growth without effects on self-flocculation. Conversely, the self-flocculation capacity of the biomass increased at higher concentrations of macronutrients. The optimization of mineral nutrition of T. tenuis allowed a 73% increase in the final biomass level and 3.5 times higher flocculation rates.

Biomass↗

Flocculation of diatomite by methylated milk casein in seawater.

A new biodegradable flocculant was prepared from a common and inexpensive protein. Milk casein was methylated in a 0.05 M HCl methyl alcohol solution at room temperature. The methylated milk casein (MeCS), having a methylation degree of 81%, was applied to the separation or flocculation of diatomite in seawater (pH 8.1+/-0.1) at room temperature (18-23 degrees C). The flocculating ability of MeCS was evaluated by a sedimentation balance method (cumulative measurement method). The diatomite suspension was effectively flocculated by the addition of a small amount of MeCS (0.25 wt% of the diatomite weight). The results of sedimentation analysis showed that the size-frequency curve had a very sharp and high peak; thus the diatomite floc formed by MeCS had a rather uniform size. The settling velocity of diatomite floc at the appropriate MeCS dosages (0.25-2 wt% of diatomite) was about 3 x 10(-3) ms(-1).

Animals↗

Determination of the critical surface tension of wetting of minerals treated with surfactants by shear flocculation approach.

This paper contributes the shear flocculation method as a new approach to determine the critical surface tension of wetting of minerals treated with surfactants. This newly developed approach is based on the decrease of the shear flocculation of the mineral suspension, with decreasing of the surface tension of the liquids used. The solution surface tension value at which shear flocculation does not occur can be defined as the critical surface tension of wetting (gamma c) of the mineral. By using the shear flocculation method, the critical surface tensions of wetting (gamma c) for calcite and barite minerals, treated with surfactants, were obtained as 30.9 and 35.0 mN/m, respectively. These values are in good agreement with data reported previously on the same minerals obtained by the contact angle measurement and flotation methods. The chemical agents used for the treatment of calcite and barite particles were sodium oleate and sodium dodecyl sulfate, respectively.

Flocculation↗

Flocculation behaviour of model textile wastewater treated with a food grade polysaccharide.

In this study, the application of a food grade polysaccharide namely Plantago psyllium mucilage has been assessed for the removal of dyes from model textile wastewater containing golden yellow (C.I. Vat Yellow 4) and reactive black (C.I. Reactive Black 5). A series of contact time experiments were conducted to assess the system variables such as concentrations of mucilage and dyes and pH. This mucilage reduces the dye concentration by flocculation and settling. The optimal flocculant concentration required to affect flocculation is independent of dye concentration within the range examined. The dye removal obtained was influenced by the salts concentrations in the wastewater sample. The flocculation efficiency was sensitive to pH when pure aqueous solutions of dyes were used, but it was relatively unaffected by pH change when salts were added to the dye solutions. The experimental results show that the mucilage is more effective for removal of solubilised vat dye than for reactive black.

Coloring Agents↗

Study on agglutinating factors from flocculent Saccharomyces cerevisiae strains.

The lectin-like theory suggest that yeast flocculation is mediated by an aggregating lectinic factor. In this study we isolated an agglutinating factor, which corresponds to lectin, from whole cells by treating the flocculent wild-type Saccharomyces cerevisiae NCYC 625 strain and its weakly flocculent mutant [rho degrees ] with EDTA and two non-ionic surfactants (Hecameg and HTAC). The dialysed crude extracts obtained in this way agglutinated erythrocytes and this hemagglutination was specifically inhibited by mannose and mannose derivatives. However, SDS-PAGE profiles showed that the three reagents had different effects on the yeast cells. The non-ionic surfactants appeared to be the most efficient, as their extracts possessed the highest specific agglutinating activity. The products released by the wild-type strain presented a higher specific agglutinating activity than those released by the [rho degrees ] mutant. Purification of the agglutinating factor from extracts of both strains by affinity chromatography revealed two active bands of relative mass of 26 and 47 kDa on SDS-PAGE. Mass spectrometry analysis by MALDI-TOF, identified a 26 kDa band as the triose phosphate isomerase (TPI) whereas a 47 kDa band was identical to enolase. Edman degradation showed that the N-terminal sequences of these proteins were similar to TPI and enolase, respectively. The difference in the flocculation behaviour of the two strains is due to changes in the protein composition of the cell wall and in the protein structure involved in cell-cell recognition.

Agglutinins↗

Modeling of orthokinetic flocculation of Saccharomyces cerevisiae.

This study examined the flocculation behavior of two Saccharomyces cerevisiae strains expressing either Flo1 (LCC1209) genotype or NewFlo (LCC125) phenotype in a laminar flow field by measurement of the fundamental flocculation parameter, the orthokinetic capture coefficient. This orthokinetic capture coefficient was measured as a function of shear rate (5.95-223 s(-1)) and temperature (5-45 degrees C). The capture coefficients of these suspensions were directly proportional to the inverse of shear rate, and exhibited an increase as the temperature was increased to 45 degrees C. The capture coefficient of pronase-treated cells was also measured over similar shear rate and temperature range. A theory, which predicts capture coefficient values due to zymolectin interactions, was simplified from that developed by Long et al. [Biophys. J. 76: (1999) 1112]. This new modified theory uses estimates of: (1) cell wall densities of zymolectins and carbohydrate ligands; (2) cell wall collision contact area; and (3) the forward rate coefficient of binding to predict theoretical capture coefficients. A second model that involves both zymolectin interactions and DLVO forces was used to describe the phenomenon of yeast flocculation at intermediate shear ranges, to explain yeast flocculation in laminar flow.

Antigen-Antibody Reactions↗

On the doublet formation in the flocculation process of the yeast cells.

The combination of single cells to form doublets is regarded as the rate-limiting step of flocculation and requires the presence of surface proteins in active form. The process of activation of the flocculation proteins of yeast cells is described in the frame of the autocrine interaction regime (Science 224 (1984) 1312). The influence of several effectors (the cell efficiency to use sugars, the calcium content in the external medium and the probability that free cells collide each other under thermal motion conditions) on the initial rate of flocculation and on the fraction of remaining free cells in the steady state is briefly discussed in the paper. The present model offers a useful tool for further quantitative investigations in this topic. Also, it indicates qualitatively a way in which the regulation of flocculation might be controlled at the level of the expression of cell-surface activation abilities.

Calcium↗

The impact of brewing yeast cell age on fermentation performance, attenuation and flocculation.

Individual cells of the yeast Saccharomyces cerevisiae exhibit a finite replicative lifespan, which is widely believed to be a function of the number of divisions undertaken. As a consequence of ageing, yeast cells undergo constant modifications in terms of physiology, morphology and gene expression. Such characteristics play an important role in the performance of yeast during alcoholic beverage production, influencing sugar uptake, alcohol and flavour production and also the flocculation properties of the yeast strain. However, although yeast fermentation performance is strongly influenced by the condition of the yeast culture employed, until recently cell age has not been considered to be important to the process. In order to ascertain the effect of replicative cell age on fermentation performance, age synchronised populations of a lager strain were prepared using sedimentation through sucrose gradients. Each age fraction was analysed for the ability to utilise fermentable sugars and the capacity to flocculate. In addition cell wall properties associated with flocculation were determined for cells within each age fraction. Aged cells were observed to ferment more efficiently and at a higher rate than mixed aged or virgin cell cultures. Additionally, the flocculation potential and cell surface hydrophobicity of cells was observed to increase in conjunction with cell age. The mechanism of ageing and senescence in brewing yeast is a complex process, however here we demonstrate the impact of yeast cell ageing on fermentation performance.

Alcian Blue↗

Influence of flocculation and adsorption as pretreatment on the fouling of ultrafiltration and nanofiltration membranes: application with biologically treated sewage effluent.

Membrane fouling is a critical limitation on the application of membranes to wastewater reuse. This work aims to understand the fouling phenomenon which occurs in ultrafiltration (UF; 17500 molecular weight cutoff (MWCO)) and nanofiltration (NF; 250 MWCO) membranes, with and without pretreatment. For this purpose, the molecular weight (MW) distribution of the organics has been used as a parameter to characterize the influent, the permeate, and the foulant on the membrane surface. The variation of foulant concentration on the membrane due to pretreatment of the influent by flocculation and/or adsorption was investigated in detail. With the UF membrane, the peak of the MW distribution of organics in the permeate depended on the pretreatment; for example, the weight-averaged MW (Mw) of 675 without pretreatment shifted down to 314 with pretreatment. In the case of the NF membrane, the Mw of organics in the permeate was 478 (without pretreatment) and 310 (with flocculation followed by adsorption). The Mw of the organics in the foulant on the membrane surface was 513 (UF) and 192 (NF) without pretreatment and 351 (UF) and 183 (NF) after pretreatment with flocculation followed by adsorption, respectively. Without the pretreatment, the foulant concentration was higher on both membranes. The difference was more significant on the UF membrane than on the NF membrane. For both membranes, the flocculation-and-then-adsorption pretreatment proved very effective.

Adsorption↗

Orthokinetic flocculation of caseinate-stabilized emulsions: influence of calcium concentration, shear rate, and protein content.

Calcium-induced flocculation of caseinate-stabilized soybean oil-in-water emulsions in conditions of Couette flow was studied. A concentrated emulsion (20% oil, 0.5-2.0% sodium caseinate in 20 mM imidazole, pH 7) was diluted 20 times in buffer containing concentrations of CaCl(2) between 9 and 17 mM and sheared at rates between 335 and 1340 s(-)(1). The average particle size (d(43)), measured by integrated light scattering, increased in a sigmoidal manner with shearing time. An increased shear rate resulted in an increased flocculation rate, because of the increased number of collisions between particles, but a decreased value of the maximum d(43), because higher shear rates increasingly disrupted the flocs. The flocculation rate was increased by increasing the calcium concentration, indicating an increased collision efficiency. The orthokinetic stability of the emulsions was increased with increased protein content, and it is postulated that the increased surface coverage and hydrodynamic thickness of the adsorbed protein layer increased steric repulsion between droplets, so that higher calcium concentrations were necessary to induce sufficient conformational change of the proteins to allow flocculation. At high caseinate concentrations, calcium may also induce precipitation of unadsorbed caseins from the serum to the oil-water interface, thereby increasing steric repulsion and hence increasing orthokinetic stability.

Calcium↗

Influence of free protein on flocculation stability of beta-lactoglobulin stabilized oil-in-water emulsions at neutral pH and ambient temperature.

The influence of protein concentration and order of addition relative to homogenization (before or after) on the extent of droplet flocculation in oil-in-water emulsions stabilized by a globular protein was examined using laser diffraction. n-Hexadecane (10 wt%) oil-in-water emulsions (pH 7, 150 mM NaCl) stabilized by beta-lactoglobulin (beta-Lg) were prepared by three methods: (1) 4 mg/mL beta-Lg added before homogenization; (2)10 mg/mL beta-Lg added before homogenization; (3) 4 mg/mL beta-Lg added before homogenization and 6 mg/mL beta-Lg added after homogenization. Emulsion 1 contained little nonadsorbed protein (<3%) and underwent extremely rapid and extensive droplet flocculation immediately after homogenization. Emulsion 2 contained a significant fraction of nonadsorbed beta-Lg and exhibited relatively slow droplet flocculation for some hours after homogenization. Measurements on Emulsion 3 showed that the extremely rapid particle growth observed in Emulsion 1 could be arrested by adding native beta-Lg immediately after homogenization. The extent of particle growth in the three types of emulsions was highly dependent on the time that the salt was added to the emulsions, i.e., after 0 or 24 h aging. We postulate that the observed differences are due to changes in droplet surface hydrophobicity caused by differences in the packing or conformation of adsorbed proteins. Our data suggest that history effects have a strong influence on the flocculation stability of protein-stabilized emulsions, which has important implications for the formulation and production of protein stabilized oil-in-water emulsions.

Adsorption↗

Treatment of textile wastewater using a natural flocculant.

The physicochemical treatment of wastewater is of substantial interest, especially when conventional treatments by biological processes are not amenable. Among the current chemical processes used for industrial wastewater treatment, coagulation-flocculation has received a large attention for high pollutant removal efficiency. This paper summarizes the results of a textile wastewater treatment process aimed at the destruction of colour by coagulation-flocculation process and using an organic natural flocculant: tannic substances. Jar-test experiments were carried out in order to determine the optimum conditions for the removal of organic matter and color. Treatment with studied flocculent (Polysep3000) proved to be effective in a wide pH range (pH < or = 10), very effective in removing the color (96 %) and a fraction of the COD (40-50 %) from the textile wastewater. Polysep3000 is compared to Al2 (SO4)3 and FeCl3 for sludge production. Polysep3000 seems to be suitable for yielding a high removal of color (> 90%) and corresponding low volume of settled sludge.

Flocculation↗

Counterflow co-flocculation flotation for water purification.

A new method for potable water treatment was brought forward and studied in this research. The treatment process was named as counterflow co-flocculation flotation (CC-FF). Pilot experiment was conducted and the operational parameters were presented. The optimized operational conditions are as follows: the detention time is 6-11 min with hydraulic load of 9-16 m3/(m2 h); the recycle ratio should be no less than 8% while the distance between the inlet of source water and recycle water should be greater than 1200 mm. If the source water turbidity was lower than 100 NTU, 0.12-0.35 mmol/L Al dosage is enough to maintain efficient turbidity removal. Since the flocculation and flotation processes were carried out in the same tank, this new technique has some advantages than the conventional flocculation-flotation methods. Firstly, the microbubbles released from recycle water will participate in the flocculation of suspended particles, hence the low-density but high shear-force-resistance flocci could be formed. Secondly, the microflocci or suspended particles will be functioned as 'nucleus' during the bubble formation from air-dissolved recycle water. Thirdly, in the midsection of the tank a blanket of bubble-microfloc aggregates could be formed, which will intercept the downward-flow flocci and upward-flow bubbles efficiently, thus keep the renovation and stability of the blanket.

Conservation of Natural Resources↗

Down-regulation of the expression of PKC1 and SRB1/PSA1/VIG9, two genes involved in cell wall integrity in Saccharomyces cerevisiae, causes flocculation.

The cell wall integrity determinants PKC1 and SRB1/PSA1/VIG9 of Saccharomyces cerevisiae were expressed under the control of the tightly regulated promoter pMET3. Substitution of the cell-cycle-regulated SRB1/PSA1 native promoter with pMET3 led to faster cell growth, larger cell volumes, and a twofold reduction of the steady-state SRB1/PSA1 mRNA level. In addition, the new pattern of expression of SRB1/PSA1 resulted in a dominant flocculation phenotype at all phases of batch growth. By contrast, expression of PKC1 from pMET3 increased the flocculation capacity of cells only at stationary phase. Methionine-mediated repression of either PSA1/SRB1 or PKC1 resulted in enhanced cell clumping. Cells in which both these genes had been replaced with their respective pMET3-regulated cassettes were highly flocculent under both expression and repression conditions. These results suggest that greater exposure of flocculin on the cell surface, caused by either cell wall distortion (through depletion of Pkc1p) or aberrant regulation of mannosylation (through constitutive production of Srb1p), results in an increased flocculation ability.

Cell Wall↗

The importance of surface charge and hydrophobicity for the flocculation of chain-forming brewing yeast strains and resistance of these parameters to acid washing.

The cell surface charge and hydrophobicity of the brewing yeast cell surface influences flocculation. Physiological stress, such as starvation, affects the capacity of some strains to flocculate due to the reorganisation of the cell wall and the modification of the surface physical properties. On completion of a brewery fermentation, yeast is removed from the beer, stored and inoculated (repitched) into a subsequent fermentation. Prior to repitching, brewing yeast slurries may be acid washed to remove any contaminating bacteria. This treatment has been shown to cause yeast cell surface "blistering". Acid washing treatment was used to examine the susceptibility of the physical properties of two chain-forming brewing yeast strains to stress. Although acid-washing affected the cell surface charge and hydrophobicity in both strains, the flocculation response was strain dependant. It is suggested that surface charge and the non-separation of progeny from mother cells rather than hydrophobicity influences the flocculation of chain-forming brewing yeast.

Acids↗

Cloning of a new FLO gene from the flocculating Saccharomyces cerevisiae IM1-8b strain.

A flocculation conferring gene was cloned from a genomic library of the flocculating strain Saccharomyces cerevisiae IM1-8b as a 5 kb DNA fragment. The shortest DNA fragment (XbaI-XbaI) able to confer the flocculating phenotype was 3.1 kb. Southern analysis revealed that this gene was not homologous to the already reported FLO1 gene since strong hybridization signals were obtained when chromosomes IV and XII were probed with a digoxygenin-labelled fragment and no signal at all was detected for chromosome I. Partial sequencing data unequivocally ascribed the cloned fragment to chromosome XII. The gene was detected in a variety of S. cerevisiae strains regardless of their being phenotypically flocculating. This gene which, we propose as FLO2, is able to complement the flo1 mutation and is suppressed by suppressors (fsu3) that do not affect other FLO genes.

Chromosome Mapping↗