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Flocculation of Saccharomyces cerevisiae: inhibition by sugars.

Flocculation is governed by the competition between electrostatic repulsion (nonspecific interactions) and polysaccharide-protein bonds (specific interactions). In our study, the inhibition of flocculation by sugars for 12 strains of Saccharomyces cerevisiae leads us to extend the classification described in the literature and to define three groups of yeasts: flocculation mannose sensitive (MS), flocculation glucose-mannose sensitive (GMS), and flocculation mannose insensitive (MI). Only the first two groups showed specific interactions between proteins and mannans. n the MI group, the sugars tested did not inhibit flocculation. To characterize the particularities of the stereochemistry of the cell-wall proteic receptors of strains belonging to the MS and GMS groups, 31 sugars were used as inhibitor probes on two representative strains. The results show that the lectin specificity of strains belonging to the GMS group is less restricted regarding C-1 and C-2 hydroxyl groups than the lectin from strains belonging to the MS group, which interacts with all of the hydroxyl groups of mannopyranose. The two groups also differ with respect to inhibition by sugars: strains belonging to the MS group are partially inhibited whereas strains of the GMS group are completely inhibited. We observed that the presence of ethanol increases sugar fixation by strains from the MS group, but not from the GMS group. Moreover, both receptors interact with disaccharides, provided the two monomers are linked by an alpha(1-4), alpha(1-3), or alpha(1-2) bond.

Calcium↗

Flocculation of Saccharomyces cerevisiae is induced by transformation with the GAP1 gene from Kluyveromyces marxianus.

A non-flocculent strain of Saccharomyces cerevisiae was transformed with the GAP1 gene which encodes p37, a GAPDH-like protein present in the cell wall of Kluyveromyces marxianus flocculent cells. The transformed cells were characterized with respect to flocculation behaviour, morphology, growth, cell wall integrity and GAPDH activity. A flocculent phenotype was acquired by the transformed cells, showing a behaviour in respect to flocculation/deflocculation very similar to that of K. marxianus. The presence of p37 in the cell wall was assessed by immunoprecipitation of biotinylated cell wall proteins and an accumulation of p37 was evident in the cell wall of transformed cells. This result was confirmed by studies using a chimeric protein resulting from fusing the p37 with a yeast-enhanced green fluorescent protein, yEGFP. The recombinant protein was localized mainly in the cell wall of the transformed strain, although the presence of p37 in the cytosol was indicated by an increase in GAPDH activity. Calcofluor white sensitivity tests indicated that the cell wall structure is affected by the accumulation of p37. These results provided further evidence of p37 function regarding flocculation and that although lacking a N-terminal signal peptide p37 is targeted to the cell wall.

Biomass↗

Zeta potential as a measure of polyelectrolyte flocculation and the effect of polymer dosing conditions on cell removal from fermentation broth.

Characterization of flocculation for cell removal from fermentation broth via polyelectrolyte addition is commonly based on qualitative methods such as physical appearance of the floc. The use of zeta potential as a quantitative measure of floc character was evaluated as an indicator of optimal polymer addition. Zeta potential was found to increase with increasing cationic polyelectrolyte dosage, but never reached zero regardless of the total amount of polymer added, indicating flocculation occurs at least partially through a bridging type mechanism. Experiments were conducted using various polymer concentrations (25-75 g/L) and dosing methods (batch, incremental and continuous addition) that resulted in variable overall polymer requirements to achieve optimum flocculation. Zeta potential was found to be constant at optimal floc character regardless of the total amount of polymer added, polymer concentration, or method of polymer addition. Experiments with two additional types of fermentation broth also showed characteristic zeta potentials at optimal flocculation. Polymer requirements to achieve a particular floc character can vary greatly, depending on polymer dosing conditions and fermentation batch. The effect of polymer dosing conditions on the polymer requirement to obtain optimal floc character was evaluated. Polymer dosing method and calcium concentration were both found to have a significant effect (P < 0.0001) with continuous polymer addition and high calcium concentration requiring less polymer than did batch polymer addition and low calcium concentration, respectively. Polymer dosing concentration did not significantly affect polymer requirement for optimal flocculation.

Bacillus↗

The regulation of Saccharomyces cerevisiae FLO gene expression and Ca2+ -dependent flocculation by Flo8p and Mss11p.

The ability of many microorganisms to modify adhesion-related properties of their cell surface is of importance for many processes, including substrate adhesion, cell-cell adhesion, invasive growth, pathogenic behaviour and biofilm formation. In the yeast Saccharomyces cerevisiae, a group of structurally related, cell-wall associated proteins encoded by the FLO gene family are directly responsible for many of the cellular adhesion phenotypes displayed by this organism. Previous research has suggested that the differential transcription of FLO genes determines specific adhesion phenotypes. However, the transcriptional regulation of most FLO genes remains poorly understood. Here we show that the transcriptional activator Mss11p, which has previously been shown to be involved in the regulation of starch degradation, the formation of pseudohyphae and haploid invasive growth, also acts as a strong inducer of flocculation. The data indicate that Mss11p induces flocculation together with Flo8p, and that FLO1 is the dominant target gene of the two factors in this process. The deletion of MSS11 leads to a non-flocculent phenotype, and specific domains of Mss11p that are critical for the induction of flocculation are identified. The data clearly show that several essential transcription factors are shared by at least two flocculation genes that control different adhesion phenotypes.

Calcium↗

Flocculation of colloidal clay by bacterial polysaccharides: effect of macromolecule charge and structure.

The molecular mechanism of montmorillonite flocculation by bacterial polysaccharides was investigated, with special emphasis on the effect of carboxylic charges in the macromolecules on the mechanisms of interaction with the clay surface. An indirect way to quantify the energy of interaction was used, by comparing the flocculation ability of variously acidic polysaccharides. Data on tensile strength of aggregates in diluted suspension were collected by timed size measurements in the domain 0.1-600 microm, using laser diffraction. The flow behavior of settled aggregates was studied by rheology measurements. Flocculation of colloidal clay suspension by polysaccharides requires cancelling of the electrostatic repulsions by salts, which allows approach of clay surfaces close enough to be bridged by adsorbing macromolecules. The amount of acidic charges of the polysaccharides, and especially their location in the molecular structure, governs the bridging mechanism and the resulting tensile strength of the aggregates. The exposure of carboxylate groups located on side chains strongly promotes flocculation. In turn, charges located on the backbone of the polysaccharide are less accessible to interaction, and the flocculation ability of such polysaccharides is lowered. Measurements at different pH indicate that adsorption of acidic polysaccharides occurs via electrostatic interactions on the amphoteric edge surface of clay platelets, whereas neutral polysaccharides rather adsorb via weak interactions. Increased tensile strength in diluted aggregates due to strong surface interactions results in proportionally increased viscosity of the concentrated aggregates.

Aluminum Silicates↗

Influence of trivalent electrolytes on the humic colloid-borne transport of contaminant metals: competition and flocculation effects.

With the objective to assess the relevance of competitive effects in respect of the humic colloid-borne migration of actinides in case of release, the influence of Al(III) on humate complexation of La(III) as an analogue of trivalent actinides was investigated for various humic materials by using 140La as a radioactive tracer, allowing measurements in very dilute systems to simulate realistic settings. Generally, competition by aluminium is not detectable unless the metal-loading capacity of the humic colloids is nearly exhausted. For average contents of organic carbon, a threshold value of 10(-6) M Al(III) can be specified. The metal exchange turned out to be kinetically hindered. Effects on co-adsorption of La(III) and humic acid were found to be less important. Immobilization by the concomitantly induced flocculation process outweighs the role of displacement effects. Comparative studies on complexation and flocculation of humic acid with Al(III), Ga(III), In(III), Sc(III), Y(III), and La(III) were undertaken in order to evaluate the influence of specific properties apart from ion charge and to characterize the mechanism of flocculation. In spite of considerable variations in the binding affinities among these metals, it can be inferred that the possibility of significant competitive effects in natural aquatic systems is confined to Al(III). Complex stabilities and flocculation efficiencies proved to be interrelated. Precipitation is thus attributed to homocoagulation of humic colloids induced by charge compensation, which is further supported by flocculation experiments with Al(III) depending on pH, ionic strength, and humic acid concentration.

Adsorption↗

Mechanism of kaolinite and titanium dioxide flocculation using chitosan--assistance by fulvic acids?

Flocculation of kaolinite using chitosan alone as flocculant has been reported to be difficult. Detailed investigations in our laboratory have shown that kaolinite can be easily removed from aqueous suspensions by flocculation and settling using chitosan, provided the suspension medium contains traces of dissolved humic substances. The flocculation is also extremely sensitive to the pH of the suspension. Removal of suspended titanium dioxide particles in water by flocculation using chitosan in presence of humic acids is also reported for the first time. A plausible mechanism explaining these observations is proposed in this paper.

Benzopyrans↗

Comparative study of humic acids flocculation with chitosan hydrochloride and chitosan glutamate.

Flocculation of dissolved humic acids (HA) and their complexes with metal ions and organic pollutants is a crucial step in drinking water preparation due to significant undesirable effects of humic compounds on water quality. Constant demand for cheap, effective and environmentally friendly flocculants brings about the interest to natural cationic polymers which can be used to neutralize negative charge of humic colloids through polyelectrolyte complexes (PEC) formation. In this work, the interaction of HA with two types of soluble chitosan salts (hydrochloride and glutamate) at different pH and polymer ratios was studied with a focus on electrical charge of PEC formed and effectiveness of color removal in the flocculation process. For both chitosan forms flocculation window was found at pH close to the PEC isoelectric point (pI) in a wide concentration range; however, lower flocculant dose was required when chitosan glutamate was used.

Biocompatible Materials↗

Flocculation of kaolinite suspensions in water by chitosan.

Flocculation of kaolinite suspensions in water using chitosan was studied in the pH range 5-9 and the turbidity ranging from 10 to 160 NTU. Chitosan, in presence of trace quantities of a substance present in aqueous soil extracts, effectively reduces turbidity due to kaolinite by flocculation and settling. Flocculation efficiency is very sensitive to pH, and reaches a maximum at pH 7.5. The optimal chitosan concentration required to effect flocculation is independent of kaolinite concentration within the range examined. Flocculation is faster at higher concentrations of kaolinite and the flocs are large and settle rapidly.

Aluminum Silicates↗

Flocculation of river silt using chitosan.

Flocculation of silt in river water using chitosan was studied in the pH range 4-9, and suspended solid concentrations in the range 20-80 mg/L. Chitosan effectively reduces turbidity due to silt by flocculation and settling. Flocculation efficiency is very sensitive to pH, and reaches a maximum at pH 7. The optimal chitosan concentration required to effect flocculation is 0.5 mg/L and is independent of silt concentration within the range examined. Restabilisation of the suspension is observed at higher concentrations of chitosan, and the amount required for restabilisation increases with increasing concentration of suspended solids. Flocculation is faster at higher concentrations of silt and the flocs are large and fibrous.

Chelating Agents↗

Acquisition of flocculation phenotype by Kluyveromyces marxianus when overexpressing GAP1 gene encoding an isoform of glyceraldehyde-3-phosphate dehydrogenase.

The use of flocculating yeast strains has been considered as a convenient approach to obtain high cell densities in bioreactors with increasing productivity in continuous operations. In Kluyveromyces marxianus ATTC 10022, the GAP1 gene encodes an isoform of glyceraldehyde-3-phosphate dehydrogenase-p37-that is accumulated in the cell wall and is involved in flocculation. To test the use of p37 as a tool for engineering Kluyveromyces cells to display a flocculation phenotype, K. marxianus CCT 3172 was transformed with an expression vector containing GAP1. This vector is based on the pY37 previously described, harbouring a S11 Kluyveromyces origin of replication, and the expression of GAP1 is under the control of GAL1. Kluyveromyces cells overexpressing GAP1 acquired a flocculent phenotype together with the accumulation of p37 in the cell wall. The results support the use of GAP1 gene as a molecular tool for inducing flocculation.

Blotting, Northern↗

Latex particles with thermo-flocculation and magnetic properties for immobilization of alpha-chymotrypsin.

Core-shell-type latex particles composed of styrene, N-isopropylacrylamide (NIPAAm), and N-acryloxysuccinimide (NAS) were synthesized by surfactant-free emulsion polymerization. The latex particles show thermo-flocculation behavior due to the presence of temperature-sensitive monomer NIPAAm and could be used for immobilization of alpha-chymotrypsin through covalent bonding with the reactive ester groups of NAS. Enzyme recycle could be accomplished in this immobilized enzyme system by sedimentation of the thermo-flocculated latex particles in 20 min at 30 degrees C by raising the salt (NaCl) concentration to 0.5 M. To further enhance the sedimentation rate, ultrafine magnetite particles were prepared and included during polymerization to produce magnetic temperature-sensitive latex particles (MTLP), which could be recovered 6 times faster after thermo-flocculation by applying a low magnetic field. However, a higher salt concentration was necessary to flocculate the MTLP under the same condition as a result of its increased surface hydrophilicity, which originates from different polymerization conditions and the incorporation of magnetite. The immobilized enzyme shows high activity even against macromolecular substrates (hemoglobin and casein) owing to limited diffusion resistance, with full activity retention for nonmagnetic latex but one-half reduction in activity if the magnetic property was introduced. Optimal enzyme immobilization pH and enzyme loading were determined, and properties of the immobilized enzyme were characterized. The immobilized enzyme was used in 10 repeated batch hydrolyses of casein with successive flocculation/dispersion cycles and showed less than 15% activity decrease at the end. Overall, introducing the magnetic property to the latex could effectively enhance the solid-liquid separation rate after thermo-flocculation and maintain enzyme activity after repeated use but adversely influence the activity of the immobilized enzyme.

Chymotrypsin↗

Optimization of coagulation-flocculation process for palm oil mill effluent using response surface methodology.

The coagulation-flocculation process incorporated with membrane separation technology will become a new approach for palm oil mill effluent (POME) treatment as well as water reclamation and reuse. In our current research, a membrane pilot plant has been used for POME treatment where the coagulation-flocculation process plays an important role as a pretreatment process for the mitigation of membrane fouling problems. The pretreated POME with low turbidity values and high water recovery are the main objectives to be achieved through the coagulation-flocculation process. Therefore, treatment optimization to serve these purposes was performed using jar tests and applying a response surface methodology (RSM) to the results. A 2(3) full-factorial central composite design (CCD) was chosen to explain the effect and interaction of three factors: coagulant dosage, flocculent dosage, and pH. The CCD is successfully demonstrated to efficiently determine the optimized parameters, where 78% of water recovery with a 20 NTU turbidity value can be obtained at the optimum value of coagulant dosage, flocculent dosage, and pH at 15 000 mg/L, 300 mg/L, and 6, respectively.

Facility Design and Construction↗

Impact of protein surface denaturation on droplet flocculation in hexadecane oil-in-water emulsions stabilized by beta-lactoglobulin.

The influence of globular protein denaturation after adsorption to the surface of hydrocarbon droplets on flocculation in oil-in-water emulsions was examined. n-Hexadecane oil-in-water emulsions (pH 7.0) stabilized by beta-lactoglobulin (1-wt % beta-Lg) were prepared by high-pressure valve homogenization. NaCl (0-150 mM) was added to these emulsions immediately after homogenization, and the evolution of the mean particle diameter (d) and particle size distribution (PSD) was measured by laser diffraction during storage at 30 degrees C for 48 h. No change in d or PSD was observed in the absence of added salt, which indicated that these emulsions were stable to flocculation. When 150 mM NaCl was added to emulsions immediately after homogenization, d increased rapidly during the following few hours until it reached a plateau value, while the PSD changed from monomodal to bimodal. Addition of N-ethylmaleimide, a sulfhydryl blocking agent, to the emulsions immediately after homogenization prevented (at 20 mM NaCl) or appreciably retarded (at 150 mM NaCl) droplet flocculation. These data suggests that protein unfolding occurred at the droplet interface, which increased the hydrophobic attraction and disulfide bond formation between droplets. In the absence of added salt, the electrostatic repulsion between droplets was sufficient to prevent flocculation, but in the presence of sufficient salt, the attractive interactions dominated, and flocculation occurred.

Alkanes↗

Influence of iota-carrageenan on droplet flocculation of beta-lactoglobulin-stabilized oil-in-water emulsions during thermal processing.

The influence of thermal processing on droplet flocculation in oil-in-water emulsions stabilized by either beta-lactoglobulin (primary emulsions) or beta-lactoglobulin-iota-carrageenan (secondary emulsions) at pH 6 has been investigated. In the absence of salt, the zeta-potential of the primary emulsion was less negative (-40 mV) than that of the secondary emulsion (-55 mV) due to adsorption of anionic iota-carrageenan to the anionic beta-Lg-coated droplet surfaces. The zeta-potential and mean diameter (d(43) approximately 0.3 microm) of droplets in primary and secondary emulsions did not change after storage at temperatures ranging from 30 to 90 degrees C. In the presence of 150 mM NaCl, the zeta-potential of the primary emulsion was much less negative (-27 mV) than that of the secondary emulsion (-50 mV), suggesting that the latter was less influenced by electrostatic screening effects. The zeta-potential of the primary emulsions did not change after storage at elevated temperatures (30-90 degrees C). The zeta-potential of the secondary emulsions became less negative, and the aqueous phase iota-carrageenan concentration increased at storage temperatures exceeding 50 degrees C, indicating iota-carrageenan desorbed from the beta-Lg-coated droplets. In the primary emulsions, appreciable droplet flocculation (d(43) approximately 8 microm) occurred at temperatures below the thermal denaturation temperature (T(m)) of the adsorbed proteins due to surface denaturation, while more extensive flocculation (d(43) > 20 microm) occurred above T(m) due to thermal denaturation. In the secondary emulsions, the extent of droplet flocculation below T(m) was reduced substantially (d(43) approximately 0.8 microm), which was attributed to the ability of adsorbed carrageenan to increase droplet-droplet repulsion. However, extensive droplet flocculation was observed above T(m) because carrageenan desorbed from the droplet surfaces. Differential scanning calorimetry showed that iota-carrageenan and beta-Lg interacted strongly in aqueous solutions containing 0 mM NaCl, but not in those containing 150 mM NaCl, presumably because salt weakened the electrostatic attraction between the molecules.

Calorimetry, Differential Scanning↗

Depletion-flocculation in oil-in-water emulsions using fibrillar protein assemblies.

This paper shows that low concentrations of beta-lactoglobulin fibrils can induce depletion-flocculation in beta-lactoglobulin-stabilized oil-in-water emulsions. The minimum required fibril concentration for flocculation was determined experimentally for fibril lengths of about 3 and 0.1 microm. The minimum fibril concentration for flocculation is two orders of magnitude higher for the short fibrils than for the long ones. These experimental results correspond well with a theoretical prediction based on a model of spinodal decomposition. In addition, rheological measurements were performed, verifying that flocculation was induced by a depletion mechanism. The results of this study show that the minimum concentration required for depletion-flocculation can be tuned by varying the length of the fibrils.

Emulsions↗

Evaluation of three flocculation methods for the purification of Cryptosporidium parvum oocysts from water samples.

AIMS: Evaluation of three flocculation methods for the purification of Cryptosporidium parvum oocysts from tap water. METHODS AND RESULTS: Ferric sulphate, aluminium sulphate and calcium carbonate were compared for their recovery efficiency of C. parvum oocysts from tap water. Lower mean recovery was achieved by calcium carbonate (38.8%) compared with ferric sulphate (61.5%) and aluminium sulphate (58.1%) for the recovery of 2.5 x 10(5) oocysts l(-1); 2.5 oocysts l(-1) and 1 oocyst l(-1) were adequately purified using ferric sulphate flocculation. In vitro excystation experiments showed that ferric sulphate flocculation does not markedly reduce the viability of oocysts. CONCLUSIONS: Ferric sulphate flocculation is a simple and effective tool for the purification of C. parvum oocysts from tap water. SIGNIFICANCE AND IMPACT OF THE STUDY: The high recovery rates and low impact on oocyst viability provided by ferric sulphate flocculation might be useful for the detection of Cryptosporidium oocysts in environmental water samples.

Alum Compounds↗

Application of synthetic poly(DADM) flocculants for dye wastewater treatment.

Poly(DADM) flocculants were synthesized and applied for the removal of color, turbidity and organic materials from dye wastewater. The effect of poly(DADM) on color removal was investigated by comparing two treatments: (i) alum alone and (ii) alum/poly(DADM) in combination. The effects of poly(DADM) flocculant, poly(DADM) concentration, poly(DADM) types and pH on the removal efficiency of coloring materials were investigated. Poly(DADM) flocculants were highly efficient in the removal of color and turbidity from dye wastewater. Compared to treatment with alum alone, the addition of a small amount of poly(DADM) (25 mg l(-1)) reduced alum dosage by 50% while improving color removal efficiency. Highly efficient color removal was obtained in wide pH ranges by adding poly(DADM) as a flocculant. Our results indicate that the use of poly(DADM) flocculant is cost effective in dye wastewater treatment by reducing sludge production and the dosage of inorganic coagulant. Effects of zeta potential were also investigated.

Coloring Agents↗