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PubMed1996

Flocculation onset, growth phase, and genealogical age in Saccharomyces cerevisiae.

Flocculation onset, the time during the fermentative cycle at which the strains of Saccharomyces cerevisiae become flocculent, is an important factor in the brewing industry. The flocculation ability of Flo 1 phenotype (strain NCYC 869) remained practically unchanged throughout the growth and seems to be insensitive to the presence of nutrients of the culture medium. On the contrary, the flocculation of NewFlo phenotype (strain NCYC 1195) exhibited a cyclic behaviour. It was found that the loss of flocculation in the early growth was the result of two combined effects: the dismantling of the flocculation mechanism of the cells coming from the inoculum and the nonflocculent state of the new cells produced after growth has started. The onset of flocculation of strain NCYC 1195 in the cultural conditions used in this work coincided with the end of the exponential growth, when the minimum glucose level in the culture medium was attained. It was demonstrated that it is possible to manipulate the flocculation onset by changing the initial glucose concentration in the culture medium.

Culture Media↗

PubMed2003

A randomized controlled trial of household-based flocculant-disinfectant drinking water treatment for diarrhea prevention in rural Guatemala.

We conducted a study to determine if use of a new flocculant-disinfectant home water treatment reduced diarrhea. We randomly assigned 492 rural Guatemalan households to five different water treatment groups: flocculant-disinfectant, flocculant-disinfectant plus a customized vessel, bleach, bleach plus a vessel, and control. During one year of observation, residents of control households had 4.31 episodes of diarrhea per 100 person-weeks, whereas the incidence of diarrhea was 24% lower among residents of households receiving flocculant-disinfectant, 29% lower among those receiving flocculant-disinfectant plus vessel, 25% lower among those receiving bleach, and 12% lower among households receiving bleach plus vessel. In unannounced evaluations of home drinking water, free chlorine was detected in samples from 27% of flocculant-disinfectant households, 35% of flocculant-disinfectant plus vessel households, 35% of bleach households, and 43% of bleach plus vessel households. In a setting where diarrhea was a leading cause of death, intermittent use of home water treatment with flocculant-disinfectant decreased the incidence of diarrhea.

Child, Preschool↗

PubMed1997

Ultrasonic Monitoring of Oil-in-Water Emulsions Undergoing Depletion Flocculation

The paper describes a three-part experiment on flocculating emulsions in order to determine whether ultrasonic spectroscopy could be used to detect and characterize the flocculation process. Three emulsions which represented unflocculated, partially flocculated, and fully flocculated systems were prepared. They were characterized independently using direct microscopic examination and their creaming behavior. Ultrasonic spectroscopic measurements of the flocculating emulsions showed large changes during the flocculation process, which were not readily modeled using single particle scattering theory. The ultrasonic properties of the unflocculated emulsion did not change during the measurement period. The results demonstrate that ultrasonic techniques are capable of detecting the flocculation process. There is clear potential to use ultrasonic techniques to characterize flocculated structures, but more work is needed on the theoretical analysis of the interaction of ultrasound with particle aggregates. Copyright 1997Academic Press

Journal Article↗

PubMed1998

Effect of Ionic Strength on the Initial Dynamics of Flocculation of Polystyrene Latex with Polyelectrolyte.

The effect of ionic strength on the kinetics of flocculation of polystyrene latex (PSL) particles induced by addition of excess polyelectrolyte was studied. The flocculation experiment was performed in the standardized mixing flow generated by using an end-over-end rotation apparatus and by changing the ionic strength and polyelectrolyte concentration. We focused our attention on the initial process of flocculation, which is characterized by a remarkably high rate of flocculation (the first stage) followed by an abrupt decrease in the flocculation rate (the second stage). The combined measurements of the rate of flocculation and the electrophoretic mobility as a function of mixing steps revealed a transient state of polyelectrolyte adsorbing on the bare surface of PSL particles. Under the conditions of low ionic strength, the picture is consistent with that of kinetically controlled adsorption of polyelectrolyte, while, under high ionic strength, the evidence of kinetically controlled adsorption disappeared. However, even in the latter, the rate of flocculation in the first stage was a function of polyelectrolyte concentration. This is consistent with a picture of nonequilibrium flocculation. A crossover with respect to ionic strength can be ascribed to an increase in flexibility of polyelectrolytes at colloidal surfaces with an increase in ionic strength. Copyright 1998 Academic Press.

Journal Article↗

PubMed2000

Size Dependence of Protein-Induced Flocculation of Phosphatidylcholine Liposomes.

The adsorption of lysozyme and cytochrome C on phosphatidylcholine liposomes essentially changes the physical properties of the phospholipid membranes and under certain circumstances greatly affects the stability of the colloid dispersion by inducing bridging liposome flocculation. This study was designed to examine experimentally the influence of liposome size on two kinetic parameters of the flocculation, its rate constant and activation energy. As the liposome radius increased in the range 50-500 nm, the activation energy tended to decrease, resulting in an increased flocculation rate, except for the flocculation of 400-nm liposomes, which was greatly impeded. The pronounced influence of the liposome size on the flocculation rate constant was evident, since a well-defined minimum in the kinetic rate of flocculation of 400-nm liposomes was detected experimentally. The obtained nonlinear radius dependencies of the flocculation rates and activation energies are interpreted in terms of the bridging mechanism of the protein-induced liposome flocculation and the supplementary concept of the stability of thin liquid films formed between approaching protein-adsorbed liposomes. Copyright 2000 Academic Press.

Journal Article↗

PubMed2002

Kinetics of adsorption of high molecular weight anionic polyacrylamide onto kaolinite: the flocculation process.

The adsorption kinetics of anionic polyacrylamide flocculant onto kaolinite clay are examined as a function of flocculant dosage and pH. Special attention has been given to the flocculation effect during the adsorption process and the resulting inhibition of further adsorption. At pH 8.5 the adsorption capacity of anionic polyacrylamide on kaolinite is low while at pH 4.5, the adsorption capacity increases. Flocculant adsorption has been shown to be related to the amount of available surface area, pH, flocculant dosage, and the resulting floc strength, which controls the rate of new surface area exposure and hence the continuation of further adsorption. At both pH 4.5 and pH 8.5, complete adsorption is achieved at low flocculant dosages and adsorption equilibrium is achieved at high flocculant dosages after 1 day. In contrast, at intermediate flocculant dosages adsorption equilibrium is not reached over a 7-day period, due to a continuously increasing surface area.

Journal Article↗

PubMed2004

A kinetic investigation of the flocculation of alumina with polyacrylic acid.

Using a model colloidal system of alumina and polyacrylic acid (PAA), the kinetics of flocculation was investigated at low polymer concentrations and short durations (on the order of seconds). The polymer-induced flocculation processes obeyed Von Smoluchowski's bimolecular rate equation. Increases in the concentration of the polymer resulted in higher rate constants for the flocculation process. At a fixed concentration (say 50 ppb, parts per billion), the rate constant values showed a maximum value for 250,000 g mol(-1) polyacrylic acid. At this polymer concentration, calculations of the surface coverage of alumina by PAA molecules of different molecular weights show that for all the cases the coverage is nearly the same, approximately 1x10(-3), but the flocculation response and the rates are significantly different. This trend in flocculation characteristics is attributed to the critical polymer number density requirement for effective flocculation (at least partial charge neutralization and initiation of flocculation). The mechanism governing the flocculation at ultralow concentrations (50 ppb) is the synergistic effect of partial patch neutralization and bridging.

Journal Article↗

PubMed2004

Flocculation of microgel particles with sodium chloride and sodium polystyrene sulfonate as a function of temperature.

The flocculation behavior of poly(N-isopropylacrylamide) (PNIPAM) microgel particles, containing surface sulfate groups, has been studied as a function of sodium chloride [NaCl] concentration, between 0.1 and 800 mM NaCl and over the temperature range 25-60 degrees C. The critical flocculation temperature (CFT) of the particles was determined as a function of NaCl concentration. Three regions of NaCl concentration were established. First, at very low values of [NaCl] (< approximately 25 mM), no CFT value could be determined; this implies that the interparticle electrostatic repulsion is sufficient to prevent any flocculation occurring. This remains the case even at temperatures well in excess of the lower critical solution temperature for PNIPAM in solution, where the particles are essentially deswollen. Second, at intermediate [NaCl] (approximately 25-100 mM), the CFT decreased strongly with increasing [NaCl]. In this region, the electostatic forces are weakened sufficiently for the van der Waals forces to cause flocculation. Third, at higher [NaCl] (> approximately 100 mM), the electrostatic repulsion is screened out, and the CFT decreases linearly with [NaCl]. The reason for this decrease is the fact that aqueous solutions of NaCl become increasingly poorer solvent environments for PNIPAM with increasing [NaCl]. These trends are apparent also in the values determined for the hydrodynamic size of the stable PNIPAM particles as a function of [NaCl] and temperature. It is shown that the flocculation of the PNIPAM particles is consistent with a weak, reversible flocculation model. This is apparent, for example, from the fractal dimensions of the flocs (approximately 2.0), determined from the power law used to fit the time evolution of the hydrodynamic size of the flocs, and also from the estimated depth of the mimimum in the interparticle pair potential, based on the critical size of the primary particles where flocculation just begins to occur. The effect of adding sodium poly(styrene sulfonate) [PSS] to the PNIPAM dispersions, in the absence of NaCl, was also investigated. The minimum amount of PSS required to induce flocculation was found to decrease with increasing temperature.

Journal Article↗

PubMed1982

Possible mechanism for flocculation interactions governed by gene FLO1 in Saccharomyces cerevisiae.

A model is proposed for the mechanism of flocculation interactions in yeasts in which flocculent cells have a recognition factor which attaches to alpha-mannan sites on other cells. This factor may be governed by the expression of the single, dominant gene FLO1. Isogenic strains of Saccharomyces cerevisiae, differing only at FLO1 and the marker genes ade1 and trp1, were developed to examine the components involved in flocculene. Electron microscopy and concanavalin Aferritin labeling of aggregated cells showed that extensive and intense interactions between cell wall mannan layers mediated cell aggregation. The components of the mannan layer essential for flocculence were Ca2+ ions, alpha-mannan carbohydrates, and proteins. By studying the divalent cation dependence at various pH values and in the presence of competing monovalent cations, flocculation was found to be Ca2+ dependent; however, Mg2+ and Mn2+ ions substituted for Ca2+ under certain conditions. Reversible inhibition of flocculation by concanavalin A and succinylated concanavalin A implicated alpha-branched mannan carbohydrates as one essential component which alone did not determine the strain specificity of flocculence, since nonflocculent strains interacted with and competed for binding sites on flocculent cells. FLO1 may govern the expression of a proteinaceous, lectin-like activity, firmly associated with the cell walls of flocculent cells, which bind to the alpha-mannan carbohydrates of adjoining cells. It was selectively and irreversibly inhibited by proteolysis and reduction of disulfide bonds. The potential of this system as a model for the genetic and biochemical control of cell-cell interactions is discussed.

Agglutination↗

PubMed2026

Inducible flocculation in Komagataella phaffii enables enhanced biomass separation for biopharmaceutical production.

Biomass separation represents a critical bottleneck in Komagataella phaffii-based biopharmaceutical processes, as typically high cell densities of 40 - 50 % create significant operational, technical and economic challenges for harvest operations. Yeast cell aggregation (flocculation) provides a solution to accelerate cell sedimentation by increasing particle size, thus allowing to improve biomass-supernatant separation efficiency during both natural gravity settling and (continuous) centrifugation operations. This study demonstrates successful engineering of K. phaffii strains with an inducible flocculation phenotype using CRISPR/Cas9-based genome editing to integrate the Saccharomyces cerevisiae FLO1 (ScFLO1) gene under control of various regulatory elements, including methanol-inducible and derepressible promoters. Flocculation strength could be enhanced by implementing transcriptional positive feedback circuits based on the methanol-inducible AOX1 promoter. To address methanol-free production requirements, we developed alternative systems to retrofit PAOX1-based ScFLO1 expression and exploited the derepressible PDF promoter, offering broader compatibility with biopharmaceutical manufacturing facilities. Flocculating cells cultivated in a bioreactor demonstrated significantly improved sedimentation behavior, with considerably lower supernatant turbidity after short low-speed centrifugation or gravity sedimentation compared to non-flocculating controls. Crucially, cell flocculation had no negative impact on product amount and quality when expressing a multivalent NANOBODY® VHH molecule with pharmaceutical relevance. Thus, this work establishes the first genetically engineered flocculation system in K. phaffii compatible with recombinant protein production, providing the basis for an innovative approach to streamline harvest operations in biopharmaceutical processes.

Flocculation↗

PubMed2000

Rapid monitoring for the enhanced definition and control of a selective cell homogenate purification by a batch-flocculation process.

Downstream-bioprocess operations, for example, selective flocculation, are inherently variable due to fluctuations in feed material, equipment performance, and quality of additives such as flocculating agents. Due to these fluctuations in operating conditions, some form of process control is essential for reproducible and satisfactory process performance and hence, product quality. Both product (alcohol dehydrogenase) and key contaminants (RNA, protein, cell debris) within a Saccharomyces cerevisiae system were monitored in real-time adopting an at-line enzymatic reaction and rapid UV-VIS spectral-analysis technique every 135 seconds. The real-time measurements were implemented within two control configurations to regulate the batch-flocculation process according to prespecified control objectives, using the flocculant dose as the sole manipulative variable. An adaptive, model-based control arrangement was studied, which combined the rapid measurements with a process model and two model parameter-identification techniques for real-time prediction of process behavior. Based on an up-to-date mathematical description of the flocculation system, process optimization was attained and subsequent feedback control to this optimum operating set point was reproducibly demonstrated with a 92% accuracy. A simpler control configuration was also investigated adopting the cell debris concentration as the control variable. Both control arrangements resulted in superior flocculation-process performances in terms of contaminant removal, product recovery, and excess flocculant usage compared to an uncontrolled system.

Alcohol Dehydrogenase↗

PubMed2004

Flocculation of cellular suspensions by polyelectrolytes.

The regularities, kinetics and mechanisms of flocculation of Escherichia coli and B. thuringiensis var. israelensis (Bti) cellular suspensions by water-soluble polymers-and first of all cationic polyelectrolytes of different charge density and stiffness of the macromolecule chain have been investigated. The effect of the focculant dose and nature, its charge density, the hydrophobic-hydrophilic balance in macromolecule, the suspension concentration, the mode of adding the reagent, the pH and the medium composition on the degree of aggregation of cells both in perikinetic regime and in a flowing system is considered. It has been shown that the main laws of microorganism's suspension flocculation are the same as the laws of flocculation of inorganic dispersions but at the same time the first process is much more complicated because the cell-flocculant interactions are strongly affected by products of cell metabolism, components of the culture liquor, pH value, electrolyte content as well as by the changing structure of the cell surface. On the basis of complex measurements of polymer adsorption and its effect on the electrokinetic potential and degree of aggregation of cells, a conclusion is made that the aggregation of E. coli cells by flexible polyelectrolytes like polydiethylaminoethylmetacrylate and its copolymers with acrylic acid, acrylamide and vinylpyrrolidone is due to charge neutralization, while the flocculation in the presence of rigid-chain chitosan and its derivatives is caused mainly by "bridging" between cells via adsorbed macromolecules. Extraction of cells from suspension can be enhanced by combination of electroflotation and flocculation by cationic polyelectrolytes. It has been shown that dilute suspensions of Bti bacteria can be effectively flocculated and concentrated using different cationic and anionic polyelectrolytes that is necessary for its formulation and use as anti-mosquito agent.

Bacillus thuringiensis↗

PubMed2004

An accurate approach to the design of channel hydraulic flocculators.

The design of flocculators for water treatment continues to be based on the generalized and simplistic concept of mean velocity gradient within the reaction zone. This approach makes little sense for hydraulic flocculators in which the turbulence conditions are heterogenous. A theoretical, experimental and computational fluid dynamics study is presented, in which a point-to-point approach is derived, allowing variations in turbulent kinetic energy to be taken into account in determining flocculation efficiency. Results for the point-to-point calculation are compared with experimental measurements of flocculation efficiency in a full-scale model of a channel hydraulic flocculator, and an extremely good fit is obtained, demonstrating the point-to-point approach to be an accurate method of determining flocculation efficiency in channel hydraulic flocculators. A design example is presented showing how the point-to-point approach can be used in practice. It is concluded that the point-to-point approach is a much better method of design than that based on the mean velocity gradient.

Equipment Design↗

PubMed2003

Flocculation of hematite particles by a comparatively large rigid polysaccharide: schizophyllan.

We studied the flocculation kinetics and structure of hematite aggregates induced by a large rigid extracellular polysaccharide, schizophyllan. Transmission electron microscopy (TEM), atomic force microscopy (AFM), photon correlation spectroscopy (PCS), and static light scattering (SLS) were used to characterize hematite particles, schizophyllan chains, and their flocs, to follow the time evolution of floc sizes, and to determine floc fractal dimensions. A maximum flocculation rate was found at a certain schizophyllan/hematite ratio. The maximum rate was considerably smaller than the rate of diffusion-limited aggregation (DLA) of hematite particles induced by simple electrolytes. To interpret the experimental results and to reveal various factors affecting the optimal dosage, Monte Carlo simulations were performed on the flocculation of small colloidal particles by relatively long, monodisperse linear polymers. The existence of the maximum flocculation rate was confirmed by computer simulation. However, a higher optimal dosage of schizophyllan was obtained in the experiments. The difference in the optimal dosage can be attributed mostly to the higher adsorption affinity of the hematite on schizophyllan aggregates present in the initial solution and the presence of a large fraction of free polymer chains which do not participate in the flocculation process. Both experiments and computer simulations demonstrated the fractal nature of the schizophyllan-hematite flocs. The fractal dimensions of the flocs at the optimal dosage were determined. A higher fractal dimension was obtained from experiments than from computer simulations, suggesting a reconstruction of the floc structure. Finally, a two-stage flocculation mechanism for hematite particles in the presence of a relatively long schizophyllan polymer was proposed. In the first flocculation stage, the hematite particles are preferentially adsorbed onto the schizophyllan aggregates in solution. The second stage consists of the association of these reactive entities with each other and also with naked chains to form fractal flocs by a bridging mechanism, where the hematite particles play the role of ligands.

Chromatography, Gel↗

PubMed2002

Flocculation properties of pectin in various suspensions.

Pectin had a flocculating activity and its flocculating activities in various suspensions were investigated. Flocculating activity of pectin in a kaolin suspension was markedly stimulated by the addition of Al3+ and Fe3+ to the suspension. Optimum temperature for flocculating activity of pectin in the kaolin suspension was around 30 degrees C and high flocculating activity was obtained when 30 mg/l of pectin and 0.2 mM Fe3+ were added to the suspension. Other inorganic suspensions of activated carbon and acid clay were flocculated by pectin in the presence of Al3+ or Fe3+. Flocculation of organic suspensions such as cellulose and yeast by pectin occurred when 0.1-0.2 mM Fe3+ was present in the suspensions.

Aluminum↗

PubMed2003

Front-face fluorescence spectroscopy study of globular proteins in emulsions: influence of droplet flocculation.

Measurement of the intensity (I(MAX)) and/or wavelength (lambda(MAX)) of the maximum in the tryptophan (TRP) emission spectrum using front-face fluorescence spectroscopy (FFFS) can be used to provide information about the molecular environment of proteins in nondiluted emulsions. Many protein-stabilized emulsions in the food industry are flocculated, and therefore, we examined the influence of droplet flocculation on FFFS. Stock oil-in-water emulsions stabilized by bovine serum albumin were prepared by high-pressure valve homogenization (30 wt % n-hexadecane, 0.35 wt % BSA, pH 7). These emulsions were used to create model systems with different degrees of droplet flocculation, either by changing the pH, adding surfactant, or adding xanthan. Emulsions (21 wt % n-hexadecane, 0.22 wt % BSA) with different pH (5 and 7) and molar ratios of Tween 20 to BSA (R = 0-131) were prepared by dilution of the stock emulsion. As the surfactant concentration was increased, the protein was displaced from the droplet surfaces, which caused an increase in both I(MAX) and lambda(MAX), because of the change in TRP environment. The dependence of I(MAX) and lambda(MAX) on surfactant concentration followed a similar pattern in emulsions that were initially flocculated (pH 5) and nonflocculated (pH 7). Relatively small changes in FFFS emission spectra were observed in emulsions (21 wt % n-hexadecane, 0.22 wt % BSA, pH 7) with different levels of depletion flocculation induced by adding xanthan. These results suggested that droplet flocculation did not have a major impact on FFFS. This study shows that FFFS is a powerful technique for nondestructively providing information about the molecular environment of proteins in concentrated and flocculated protein-stabilized emulsions. Nevertheless, in general the suitability of the technique may also depend on protein type and the nature of the physicochemical matrix surrounding the proteins.

Chemical Phenomena↗

PubMed2005

Coagulation-flocculation pretreatment of a partially stabilized leachate from a sanitary landfill site at Alhendin (Granada, Southern Spain).

Leachate recirculation, biological, and physicochemical treatment processes are used to treat leachate landfill, but all treatment technologies seem to need a combination of two or more methods to obtain an effluent with suitable properties to eliminate environmental problems. In spite of the considerable literature focused on the coagulation-flocculation applied to landfill leachate before biological treatments, most investigations are developed to study different operating variables as leachate characteristics, type, and concentrations of chemical products applied. Two ferric coagulants (ferric chloride and Ferriclar), two aluminum coagulants (PAX-18 and SAL-8.2), an organic coagulant and flocculant (Chemifloc PA-15), several anionic flocculant (Chemifloc A05.L, A10.L, A20L, and A30.L), and a non-ionic flocculant (Chemifloc N01) were used in jar-test experiments to determine the best conditions for the removal of chemical oxygen demand (COD) and total solids (TS) from a partially stabilized leachate collected at a sanitary landfill site at Alhendín (Granada, Southern Spain). The organic coagulant-flocculant PA-15 showed highest (17%) COD and TS removal efficiencies at a concentration of 2 mL/L. All combinations of coagulants and flocculants enhanced the COD removal efficiency. The best results (more than 25% COD removal) were obtained with a combination of ferric or aluminum-based salts in combination with high molecular weight flocculants.

Flocculation↗

PubMed2001

The role of glucose in the Kluyveromyces bulgaricus flocculation phenomenon: transduction by cAMP-dependent protein kinase pathway?

Yeast flocculation appears to be dependent on several culture conditions such as nitrogen or carbon sources. In 0.2% glucose medium Kluyveromyces bulgaricus flocculation intensity is weak (10% at maximum) by comparison with flocculation in 2% glucose medium (85% maximum). Addition of glucose to K. bulgaricus in exponential growth phase in 0.2% glucose medium produced a rapid increase of the flocculation percentage during the 30 min following the addition of glucose. cAMP and 2,4-dinitrophenol showed similar effects while cAMP-dependent protein kinase (PKA) inhibitors exhibited an antagonist effect. Moreover, the induction of flocculation did not seem to imply translation of new proteins: cycloheximide had no effect, although growth was inhibited. The induction of flocculation mainly implies ATP hydrolysis for activation or secretion of galactose-specific receptors as demonstrated by treatment with NaN(3). We propose a hypothesis that involves a PKA transduction signal leading to flocculation.

2,4-Dinitrophenol↗