Integration of mechanical cell disruption and fluidised bed recovery of G3PDH from unclarified disrupted yeast: a comparative study of the performance of unshielded and polymer shielded dye-ligand chromatography systems

The development of a simplified process for the simultaneous disruption and direct selective purification of intracellular proteins from unclarified yeast disruptate has been investigated. The recovery of glyceraldehyde 3-phosphate dehydrogenase (G3PDH) from baker's yeast was selected as a potential demonstration of the generic applicability and practical feasibility of this integrated technique. The application of an adsorbent characterised by high density (UpFront steel-agarose; rho=2.65g ml(-1)) facilitated the combining of cell disruption operation (bead milling of 50% ww/v of yeast suspension at 7.2 lh(-1)) with fluidised bed dye-ligand (Cibacron Blue 3GA) adsorption operated immediately downstream of the disrupter. The adoption of a polymer shielded, dye-ligand technique advanced recovery efficiency. It was demonstrated that G3PDH could be recovered with a yield of 67.5% bound activity and a specific activity of 40.2IU mg(-1), after a single step elution with 0.15M NaCl. The generic application of this approach has been evaluated.     

Operational intensification by direct product sequestration from cell disruptates: application of a pellicular adsorbent in a mechanically integrated disruption-fluidised bed adsorption process

A novel prototype adsorbent, designed for intensified fluidised bed adsorption processes, was assembled by the emulsification coating of 4% (w/v) porous agarose upon a zirconia-silica solid core. The adsorbent, designated ZSA (particle density 1.75 g/ml, maximum pellicle depth 40 microm), was subjected to physical and biochemical comparison with the performance of two commercial adsorbents (Streamline and Macrosorb K4AX). Bed expansion qualities and hydrodynamic characteristics (N, D(axl) and B(o)) of ZSA demonstrated a marked robustness in the face of elevated velocities (up to 550 cm/h) and biomass loading (up to 30% (ww/v)) disrupted yeast cells. Cibracron Blue derivatives of the pellicular prototype (ZSA-CB), evaluated in the batch and fluidised bed recovery of glyceraldehyde 3-phosphate dehydrogenase (G3PDH) from unclarified yeast disruptates, exhibited superior capacities and adsorption/desorption performance to the commercial derivatives. These advanced physical and biochemical properties facilitated a demonstration of the direct, mechanical coupling of bead-milling and fluidised bed adsorption in a fully integrated process for the accelerated recovery of G3PDH from yeast. The generic application of such pellicular adsorbents and integrated processes to the recovery of labile, intracellular products is discussed.     

Affinity adsorption of lysozyme on a macroligand prepared with Cibacron Blue 3GA attached to yeast cells

The objective of this study was the development of affinity adsorbent particles with the appropriate characteristics to be applied in protein purification using the affinity ultrafiltration method. To prepare affinity macroligands Cibacron Blue 3GA, as a ligand molecule, was immobilized by covalent bonding onto yeast cell walls, the support material or matrix. The maximum attachment of the ligand to the matrix was 212 μmol/g (ligand dry weight/yeast dry weight). Lysozyme was selected as the protein model for the adsorption studies. Its adsorption onto the matrix without ligand and matrix with attached ligand were investigated batch-wise. The adsorption equilibrium isotherms appeared to follow a typical Langmuir isotherm. The maximum adsorption capacity (q(m)) of the Cell-Cibacron macroligand for lysozyme was 110 mg/ml of wet macroligand. The adsorbent was also employed for the separation of lysozyme from hen egg white. High purity lysozyme was obtained.     

Interaction of flavin adenine dinucleotide (FAD) with a glassy carbon electrode surface

The interaction of flavin adenine dinucleotide (FAD) with a glassy carbon electrode (GCE) surface was investigated in terms of the FAD adsorption thermodynamics and kinetics, the subsequent electroreduction mechanism, and the corresponding electron-transfer rate. The kinetics of FAD electroreduction at the GCE was found to be an adsorption-controlled process. A set of electroreduction kinetic parameters was calculated: the true number of electrons involved in the FAD reduction, n=1.76, the apparent transfer coefficient, alpha(app)=0.41, and the apparent heterogeneous electron-transfer rate constant, k(app)=1.4 s(-1). The deviation of the number of exchanged electrons from the theoretical value for the complete reduction of FAD to FADH(2) (n=2) indicates that a small portion of FAD goes to a semiquinone state during the redox process. The FAD adsorption was well described by the Langmuir adsorption isotherm. The large negative apparent Gibbs energy of adsorption (DeltaG(ads)=-39.7 +/-0.4 kJ mol(-1)) indicated a highly spontaneous and strong adsorption of FAD on the GCE. The energetics of the adsorption process was found to be independent of the electrode surface charge in the electrochemical double-layer region. The kinetics of FAD adsorption was modeled using a pseudo-first-order kinetic model.     

Coupled lactic acid fermentation and adsorption

Polyvinylpyridine (PVP) and activated carbon were evaluated for coupled lactic acid fermentation and adsorption, to prevent the product concentration from reaching inhibitory levels. The lactic acid production doubled as a result of periodical circulation of the fermentation broth through a PVP adsorption column. The adsorbent was then regenerated and the adsorbed lactate harvested, by passing 0.1 N NaOH through the column. However, each adsorption-regeneration cycle caused about 14% loss of the adsorption capacity, thus limiting the practical use of this rather expensive adsorbent. Activated carbon was found much more effective than PVP in lactic acid and lactate adsorption. The cells of Lactobacillus delbrueckii subsp. delbrueckii (LDD) also had strong tendency to adsorb on the carbon. A study was therefore conducted using an activated carbon column for simultaneous cell immobilization and lactate adsorption, in a semi-batch process with periodical medium replacement. The process produced lactate steadily at about 1.3 g l(-1)h(-1) when the replacement medium contained at least 2 g l(-1) of yeast extract. The production, however, stopped after switching to a medium without yeast extract. Active lactic acid production by LDD appeared to require yeast extract above a certain critical level (<2 g l(-1)).     

Dye-ligand membranes as selective adsorbents for rapid purification of enzymes: a case study

A new adsorbent for the selective binding of enzymes, in the form of microporous membranes carrying triazine dyes as pseudo-affinity ligand, has been implemented in the recovery of glucose-6-phosphate dehydrogenase from yeast. A detailed investigation of the process parameters has been performed. In the adsorption step, the contact time for binding G6PDH could be reduced down to 0.25 s without significant decrease of the capture efficiency. Hence, fast filtration allowed to isolate G6PDH from a dilute extract (1.6 mug G6PDH . mL(-1)), where the enzyme accounted for 1% of the proteins. The yield of the selective elution step using NADP was only 70% at best. It could be improved to near 100% by supplementing the eluent with ethylene glycol, without loss of selectivity. A Scale-up of the cross-section of the membrane by a factor of 40 allowed to purify 1140 U from 0.6 L extract from 1% to 57% purity with 82% yield, within 10 minutes. The case study presented here demonstrates the applicability of general-purpose membrane adsorbents for the purification of enzymes.     

Pilot-scale production and liquid formulation of Rhodotorula minuta, a potential biocontrol agent of mango anthracnose

AIMS: To develop a pilot-plant fermentation process for the production of the yeast Rhodotorula minuta, to be used as a biocontrol agent of mango anthracnose, using a low-cost culture medium. To develop a stable liquid formulation that preserve high viability of the yeast stored at 4 degrees C. METHODS AND RESULTS: Keeping constant the volumetric power input, a fermentation process was scaled-up from shake flasks to a 100 l bioreactor. Preharvest applications of the yeast resulted in postharvest anthracnose severity equal or lower than that observed with a chemical fungicide. Glycerol was added to the formulation as water activity reducer and xanthan gum as a viscosity-enhancing agent. Yeast initial concentration of 10(10) CFU ml(-1) resulted in 4-5 orders of magnitude decrease after 1 month of storage at 4 degrees C, whereas when it was formulated at 10(9) CFU ml(-1), the decrease was of two orders of magnitude in 6 months. CONCLUSIONS: The fermentation process was successfully scaled-up using a low-cost culture medium. Postharvest anthracnose severity could be considerably reduced using this yeast. Formulating the yeast at 10(9) CFU ml(-1) and adding glycerol (20%) and xanthan (5 g l(-1)) avoided both contamination and yeast sedimentation and it was able to preserve up to 10(7) CFU ml(-1) after 6 months at 4 degrees C. SIGNIFICANCE AND IMPACT OF THE STUDY: The yeast R. minuta is reported as a novel antagonistic micro-organism against the pathogen Colletotrichum gloeosporioides. Pilot plant production of this yeast allowed us to conduct field tests in commercial orchards during three harvest seasons. Yeast suspensions applied to mango trees reduced the fruit anthracnose severity in levels similar or better than chemical fungicides.     

A simple macroscopic model for the diffusion and adsorption kinetics of r-adenovirus

The diffusion of viruses toward cells is a limiting step of the infection process. To be modeled correctly, this step must be evaluated in combination with the adsorption of the virus to the cell surface, which is a rapid but reversible step. In this paper, the recombinant adenovirus (rAd) diffusion and its adsorption to 293S cells in suspension were both measured and modeled. First, equilibrium experiments permitted to determine the number of receptors on the surface of 293S (R(T) = 3,500 cell(-1)) and the association constant (K(A) = 1.9 x 10(11) M(-1)) for rAd on these cells based on a simple monovalent adsorption model. Non-specific binding of the virus to the cell surface was not found to be significant. Second, total virus particle degradation rates between 5.2 x 10(-3) and 4.0 x 10(-2) min(-1) were measured at 37 degrees C in culture medium, but no significant virus degradation was observed at 4 degrees C. Third, free viral particle disappearance rates from a mixed suspension of virus and cells were measured at different virus concentrations. Experimental data were compared to a phenomenological dynamic model comprising both the diffusion and the adsorption steps. The diffusion to adsorption ratio, a fitted parameter, confirmed that the contact process of a virus with a cell is indeed diffusion controlled. However, the characteristic diffusion time constants obtained, based on a reversible adsorption model, were eightfolds smaller than those reported in the literature, based on diffusion models that assume irreversible adsorption.     

Nonspecific hydrophobic interactions of a repressor protein, PhaR, with poly[(R)-3-hydroxybutyrate] film studied with a quartz crystal microbalance

The gene expression for phasins (PhaP), which are predominantly polyhydroxyalkanoates (PHAs) granule-associated proteins, is regulated by a repressor protein of PhaR through the dual binding abilities of PhaR to the target DNAs and the granules. In this study, the binding functions of PhaR to poly[(R)-3-hydroxybutyrate] (P(3HB)) were investigated quantitatively by using a quartz crystal microbalance (QCM) technique. Adsorption of PhaR onto a melt-crystallized film of P(3HB) (cr-P(3HB)) was detected as a negative frequency shift of the QCM. The time course of the frequency changes observed for PhaR adsorption was composed of a quick frequency decrease at an initial stage and a subsequent slower frequency decrease for several hours, indicating multilayered adsorption of PhaR molecules onto cr-P(3HB). The initial rapid adsorption, which corresponds to direct adsorption of PhaR molecules onto a bare surface of cr-P(3HB), was a diffusion-controlled process. Strong interactions between PhaR and cr-P(3HB) were also observed as apparently irreversible adsorption. The comparative QCM measurement of PhaR adsorption onto various types of polymers with different aliphatic chemical structures revealed that PhaR was adsorbed onto the surfaces of polymers, including cr-P(3HB), mainly by nonspecific hydrophobic interactions. These results illustrate the high affinity and low specificity for adsorption of PhaR to P(3HB).     

Chemostat study of citric acid production from glycerol by Yarrowia lipolytica

The aim of the study was to examine how the dilution rate and the chemical composition of the production medium impacts on the synthesis of citric acid by the Yarrowia lipolytica strain Wratislavia AWG7 from glycerol in a chemostat culture. The yeast Y. lipolytica Wratislavia AWG7, an acetate (acet(-)) and morphological (fil(-)) mutant, was cultured in a nitrogen- and phosphorus-limited medium at the dilution rate of 0.009-0.031h(-1) in the chemostat. Under steady-state conditions, the increase in the dilution rate was paralleled by the decrease in citric acid concentration (from 86.5 to 51.2gL(-1)), as well as by the increase in the volumetric rate (from 0.78 to 1.59gL(-1)h(-1)) and specific rate (from 0.05 to 0.18gg(-1)h(-1)) of citric acid production. The yield of the production process varied from 0.59 to 0.67gg(-1). In a 550-h continuous culture of the yeast test, at a dilution rate of 0.01h(-1), in a medium with enhanced concentrations of carbon, nitrogen and phosphorus sources, the concentration of citric acid, the concentration of biomass and the volumetric rate of citric acid production were 97.8gL(-1), 22.2gL(-1) and 0.98gL(-1)h(-1), respectively. The yield of the process decreased to 0.49gg(-1). The number of dead cells did not exceed 1% while that of the budding cells accounted for about 20%. Owing to the low content of isocitric acid and polyols, the fermentation process was characterized by a high purity. This study has produced the following finding: the double mutant Y. lipolytica AWG7 is an effective citric acid producer, with the ability to preserve its properties unchanged during the long run of the continuous chemostat process. This is a valued technological feature of such mutants.     

Stability and expression of a plasmid-containing killer toxin cDNA in batch and chemostat cultures of saccharomyces cerevisiae

A chimeric plasmid (pYT760-ADH1) containing the yeast killer toxin-immunity cDNA was transformed into a leucine-histidine mutant (AH22) and into four industrial toxin-sensitive yeasts. The chimeric plasmid was very stable and expressed toxin production (89.5 +/- 4.8% killer cells) in two of the transformed yeasts that contained the 2mu plasmid, but was lost within 10 generations from two other transformed pickle yeasts that did not contain the 2mu plasmid. It suggested that plasmid stability was dependent on the presence of the 2mu plasmid which is naturally present in some yeasts. The plasmid was extremely stable (100% killer cells) and expressed more toxin in the mutant strain AH22. The effects of dilution rate, D(h(-1)) on plasmid stability and toxin expression were studied in transformed AH22 (AH22/T3) and Montrachet 522 (522/T1) wine yeast grown in glucose-limited chemostat cultures. The results show that killer toxin production by AH22/T3 cells increased as a function of D(h(-1)) and that plasmid stability reached 100% at D >/= 0.09 +/- 0.01 h(-1). However, with Montrachet 522/T1 transformed cells, 100% plasmid stability was seen at D >/= 0.18 +/- 0.02. h(-1). We also challenged the AH22/T3 in chemostat culture (D = 0.25 h(-1)) with an equal number of untransformed cells (AH22). Transformed cells dominated the population (100%) within 8-10 h of growth, a time equivalent to two mean residence time.     

Use of a fluorescence spectroscopy technique to study the adsorption of sodium dodecylsulfonate on liposomes

The fluorescent probe 2-(p-toluidinyl)-naphthalene-6-sodium sulfonate (TNS) was used to study the surface adsorption of sublytic concentrations of the anionic surfactant sodium dodecylsulfonate (C(12)-SO(3)) on phosphatidylcholine (PC) bilayers. The number of adsorbed molecules was quantified by determination of the electrostatic potential (psi(o)) of the bilayers. The abrupt decrease in the fluorescence intensity detected even 10 s after the surfactant addition and the slight fluorescence variations with time indicated that the surfactant adsorption was very fast and almost complete. For a given number of monomers adsorbed a linear dependence between the lipid and C12-SO3 concentrations was obtained, indicating similar adsorption mechanism regardless of the surfactant concentration. Hence, a monomeric adsorption is assumed even in systems with a C12-SO3 concentration above its CMC. In addition, this linear correlation allowed us to determine the surfactant/lipid molar ratios (Re) (inversely related to the C12-SO3 ability to be adsorbed on liposomes) and the bilayer/aqueous phase coefficients (K). The fact that the lowest values for Re were always reached after 10 s of incubation corroborates the rapid kinetics of the process. The decrease in the C12-SO3 partitioning (K) when the number of surfactant molecules exceeded 15000 was possibly due to the electrostatic repulsion between the free and the adsorbed monomers, which could hinder the incorporation of new monomers on the charged surface of liposomes.     

Production of the biocontrol agent Pantoea agglomerans strain CPA-2 using commercial products and by-products

The aim of this paper was to find the nitrogen and carbon sources that provide maximum biomass production of strain CPA-2 of the biocontrol agent Pantoea agglomerans and minimum cost of media, whilst maintaining biocontrol efficacy. To reduce the cost of media, commercial products and by-products were tested. P. agglomerans can be produced using a combination of nitrogen sources such as yeast extract (5 g l(-1)) and dry beer yeast (10 g l(-1)) with inexpensive carbohydrates such as sucrose (10 g l(-1)) and molasses (20 g l(-1)), respectively, maintaining the efficacy of the biocontrol agent against Penicillium digitatum and P. italicum on oranges. The results obtained in this study could be used to provide a reliable basis for a scale-up of this fermentation process to an industrial level.     

Adsorption mechanism of cadmium on juniper bark and wood

In this study the capacity of sorbents prepared from juniper wood (JW) and bark (JB) to adsorb cadmium (Cd) from aqueous solutions at different pH values was compared. Adsorption behavior was characterized through adsorption kinetics, adsorption isotherms, and adsorption edge experiments. Results from kinetics and isotherm experiments showed that JB (76.3-91.6 micromol Cdg(-1) substrate) had 3-4 times higher adsorption capacity for Cd than JW (24.8-28.3 micromol Cdg(-1)). In addition to higher capacity, JB exhibited a higher strength of adsorption (45.3 versus 9.1 Lmmol(-1)) and faster uptake kinetics (0.0119 versus 0.0083 g micromol(-1)min(-1)) compared to JW. For both these adsorbents, increasing Cd adsorption with increasing solution pH in the range of 2-6 suggests that surface carboxyl groups (RCOOH) might be involved in interaction with Cd. Diffuse reflectance infrared Fourier transform (DRIFT) spectra showed that the surface concentration of carboxyl groups was higher on JB compared to JW. The ratio of Ca released to Cd adsorbed was 1.04 and 0.78 for JB and JW, respectively, indicating that Ca-Cd ion-exchange was the primary mechanism involved. The higher Ca content in JB (15 times more) and the surface RCOOH concentration (2.5 times more) can be attributed to the observed differences in Cd adsorption behavior between the two lignocellulosic adsorbents.     

Dosimetric assessment of radon in a vegetable system

An alpha-dose calculation due to radon uptake in anthers of Tradescantia, clone 4430, has been performed. Probability distribution density of the dose in the pollen mother cells was calculated by means of a model that simulates the interaction of separate alpha-particles with these cells. It is shown that alpha-radiation from either radon or its decay products surrounding the buds does not reach pollen mother cells because of the short-range alpha-particles. However, it is suggested that radon diffuses through the gap structure of the bud to the anther from which a radon-gas adsorption process takes place. Absorbed-dose calculations in the anther are discussed as well as their relationship to the experimental results of micronucleus induction in pollen mother cells. The radon concentration interval used (0.85 kBq m(-3)-98.16 kBq m(-3)) is equivalent to the exposure to an average environmental radon concentration (40 Bq m(-3)) for 2.3 months or 22.1 years, respectively. The lowest radon concentration to induce micronuclei was 12.1 kBq m(-3), which is 15 times in excess of that adopted for old buildings in Canada.     

Decolorization of azo dye using PVA-immobilized microorganisms

A microbial consortium having a high capacity for rapid decolorization of azo dye (RED RBN) was immobilized by a phosphorylated polyvinyl alcohol (PVA) gel. The immobilized-cell beads exhibited a color removal capability of 75%, even at a high concentration of RED RBN (500 mg l(-1)) within 12 h using flask culture. The continuous operation was conducted at a hydraulic retention time (HRT) of 5-20 h in which the dye loading rate ranged from 240 to 60 mg dye h(-1). A removal efficiency exceeding 90% was obtained at the HRT higher than 10 h. No recognizable destruction of bead appearance was observed in the 6-month operation. Examination of the mechanism of the decolorization process by cell beads indicated that it proceeded primarily by biological decolorization associated with partial adsorption of the dye onto the entrapped cells and gel matrix. Microscopic observation revealed that the microbial consortium contained in the gel beads was at least made up of three kinds of bacterial species. From the economical viewpoint, alternative cheaper nitrogen sources such as fish meal, soybean meal, pharmamedia and vita yeast powder were examined.     

Expression of the Aspergillus niger glucose oxidase gene in Saccharomyces cerevisiae and its potential applications in wine production

There is a growing consumer demand for wines containing lower levels of alcohol and chemical preservatives. The objectives of this study were to express the Aspergillus niger gene encoding a glucose oxidase (GOX; beta- d-glucose:oxygen oxidoreductase, EC 1.1.3.4) in Saccharomyces cerevisiae and to evaluate the transformants for lower alcohol production and inhibition of wine spoilage organisms, such as acetic acid bacteria and lactic acid bacteria, during fermentation. The A. niger structural glucose oxidase (gox) gene was cloned into an integration vector (YIp5) containing the yeast mating pheromone alpha-factor secretion signal (MFalpha1(S)) and the phosphoglycerate-kinase-1 gene promoter (PGK1(P)) and terminator (PGK1(T)). The PGK1(P)- MFalpha1(S)- gox- PGK1(T) cassette (designated GOX1) was introduced into a laboratory strain (Sigma1278) of S. cerevisiae. Yeast transformants were analysed for the production of biologically active glucose oxidase on selective agar plates and in liquid assays. The results indicated that the recombinant glucose oxidase was active and was produced beginning early in the exponential growth phase, leading to a stable level in the stationary phase. The yeast transformants also displayed antimicrobial activity in a plate assay against lactic acid bacteria and acetic acid bacteria. This might be explained by the fact that a final product of the GOX enzymatic reaction is hydrogen peroxide, a known antimicrobial agent. Microvinification with the laboratory yeast transformants resulted in wines containing 1.8-2.0% less alcohol. This was probably due to the production of d-glucono-delta-lactone and gluconic acid from glucose by GOX. These results pave the way for the development of wine yeast starter culture strains for the production of wine with reduced levels of chemical preservatives and alcohol.     

Exopolysaccharides produced by mixed culture of yeast Rhodotorula rubra GED10 and yogurt bacteria (Streptococcus thermophilus 13a + Lactobacillus bulgaricus 2-11)

AIMs: The studies of the production of exopolysaccharides by lactose-negative yeast and a yogurt starter co-cultivated in a natural substrate containing lactose may be considered of interest because they reveal the possibilities for high-efficiency synthesis of biopolymers by mixed cultivation. METHODS AND RESULTS: The mixed culture Rhodotorula rubra GED10 + (Streptococcus thermophilus 13a + Lactobacillus bulgaricus 2-11) was cultivated in cheese whey ultrafiltrate (WU) (44.0 g lactose l(-1)) at initial pH 6.0, 28 degrees C, under intensive aeration (air-flow rate 1.0 l l(-1) min(-1), agitation 220 rev min(-1)) in a MBR AG fermentor. The mixed culture manifested the highest activity for synthesis of exopolysaccharides (19.3 g l(-1)) and cell mass (21.0 g l(-1)) at the 84th hour. The yogurt starter synthesized neutral exopolysaccharides, while the mixed culture yeast + yogurt starter produced acidic exopolysaccharides containing uronic acid (6%). The neutral sugar composition was identified as mannose, glucose, galactose, xylose and arabinose. Mannose dominated in the polymer composition (83%) that was produced only by the yeast (97%). CONCLUSIONS: Lactose in the WU can be effectively utilized by a co-culture of lactose-negative yeast-yogurt starter for synthesis of exopolysaccharides. SIGNIFICANCE AND IMPACT OF THE STUDY: The present findings propose an alternative use of WU as a cost-effective carbohydrate substrate, and suggest that the lactose-negative yeast Rhodotorula rubra can have industrial application as producers of exopolysaccharides.     

Basic dye adsorption onto an agro-based waste material--sesame hull (Sesamum indicum L.)

The aim of this project was to establish an economical and environmentally benign biotechnology for removing methylene blue (MB) from wastewater. The adsorption process of MB onto abandoned sesame hull (Sesamum indicum L.) (SH) was investigated in a batch system. The results showed that a wide range of pH (3.54-10.50) was favorable for the adsorption of MB onto SH. The Langmuir model displayed the best fit for the isothermal data. The exothermic adsorption process fits a pseudo-second-order kinetic model. The maximum monolayer adsorption capacity (359.88 mg g(-1)) was higher than most previously investigated low-cost bioadsorbents (e.g., peanut hull, wheat straw, etc.). This study indicated that sesame hull is a promising, unconventional, affordable and environmentally friendly bio-measure that is easily deployed for removing high levels of MB from wastewater.