Copper Uptake by Ryegrass Seedlings; Contribution of Cell Wall Adsorption

Net copper uptake by cellulose discs, isolated root cell walls,and by live and dead roots of whole ryegrass seedlings, werestudied using ⁶⁴Cu as a tracer. Uptake by cellulose discs stoppedafter around 10 h while uptake by isolated root cell walls continuedfor up to 50 h. An initial fast phase of uptake consisting predominantlyof cell wall adsorption was similar in live and dead tissuefor up to 19 h. A slower phase of uptake continued for up to50 h, greater in live than in dead tissue, the slower phaseof uptake in live tissue consisting of both a living and a deadcomponent. Based on these results, an alternative to the desorptionmethod for estimating the apoplastic contribution to total copperuptake is presented. Time-course studies with seedlings givena variety of growing solution/uptake solution regimes, and therelationship between copper uptake and external copper concentration,for short (4.8 h) and long (42.4 h) term uptakes, suggest thatdiffering contributions of cell wall adsorption and symplasmicabsorption may be responsible for differing effects of externalcopper concentration on uptake being expressed by the same tissue.Water flux had little effect on total uptake of copper althougha possible effect on absorption could not be ruled out. Key words: Copper uptake, cell wall adsorption, ryegrass seedlings     

产朊假丝酵母细胞壁对铜离子吸附机理研究

比较了产朊假丝酵母细胞与分离纯化的细胞壁对铜离子吸附能力。观察铜离子浓度、温度和pH值对产朊假丝酵母吸附铜离子的影响，探讨细胞壁在酵母吸附重金属离子过程中的作用机理。结果表明，细胞壁是酵母吸附重金属离子的主要部位。细胞壁的蛋白酶酶解实验证明，对胰蛋白酶不敏感的细胞壁嵌合蛋白是铜离子吸附的主要位点。     

Diffusion coefficients of glucose and ethanol in cell-free and cell-occupied calcium alginate membranes

The diffusivities of glucose and ethanol in cell-free and cell-occupied membranes of calcium alginate were measured in a diffusion cell. The lag time analysis was used. Diffusivities decreased with increasing alginate concentration and were comparable with those in water for a 2% alginate membrane. Glucose and ethanol concentrations had no effect on the respective diffusion coefficients. The ratio of ethanol diffusivity to glucose diffusivity in 2 and 4% alginate agreed closely with the inverse ratio of the hydrodynamic raii for the two molecules in water, indicating that the hydrodynamic theory of diffusion in liquids may be applicable to diffusion in dilute alginate gels. Also, the presence of 20% dead yeast cells had no effect on the diffusivities. The data reported can be used to study reaction and diffusion in immobilized cell reactors and cell physiology under immobilized conditions.     

Physicochemical parameters involved in the interaction of Saccharomyces cerevisiae cells with ion-exchange adsorbents in expanded bed chromatography

Expanded bed adsorption (EBA) is an interesting primary technology allowing the adsorption of target proteins from unclarified feedstock in order to combine separation, concentration, and purification steps. However, interactions between cells and adsorbent beads during the EBA process can strongly reduce the performance of the separation. So, to minimize these interactions, the mechanisms of cell adsorption on the support were investigated. Adsorption kinetics of the baker's yeast Saccharomyces cerevisiae on the anion exchanger Q Hyper Z were directly performed under real EBA operating conditions, in a lab-scale UpFront 10 column. The yeast was marketed either as rod-shaped pellets (type I yeast) or as spherical pellets (type II yeast). For both types, a complete series of experiments for determining the adsorption profile versus time was performed, varying the superficial velocity or the pH. In parallel, the surface physicochemical properties of the cells (surface charge and electron-donor and electron-acceptor components) and of the support were determined. First of all, whatever the yeast types, the relation between cell adsorption and bed expansion has been highlighted, demonstrating the important role of hydrodynamic. However, for the type II yeast cells, adsorption increased dramatically, compared to the type I, even though it was shown that both types exhibited the same surface charge. In fact, there were strong differences in the Lewis acidic and basic components of the two yeasts. These differences explain the variable affinity toward the support, which was characterized by a strong electron-donor and a weak electron-acceptor component. These observed behaviors agreed with the colloidal theory. This work demonstrates that all kinds of interaction between the cells and the support (electrostatic, Lifshitz-van der Waals, acid/base) have to be taken into account together with hydrodynamic characteristics inside the bed.     

A study of the influence of yeast cell debris on protein and alpha-glucosidase adsorption at various zones within the expanded bed using in-bed sampling

Expanded bed adsorption chromatography is used to capture products directly from unclarified feedstocks, thus combining solid-liquid separation, product concentration and preliminary purification into a single step. However, when non-specific ion-exchangers are used as the adsorbent in the expanded bed, there is the possibility that electrostatic interactions of cells or cell debris with the adsorbent may interfere with the adsorption of soluble products. These interactions depend on the particle size of the cell debris and its surface charge, which in turn depend on the extent of disruption used to release the intracellular products. The interactions occurring during expanded bed adsorption between the anionic ion-exchanger STREAMLINE DEAE and particulate yeast homogenates obtained by high pressure homogenisation at different intensities of disruption achieved by operating at different pressures were studied, while maintaining all other parameters constant. In-bed sampling from the expanded bed using ports fitted up the height of expanded bed was used to study the retention of yeast cells and cell debris within the bed and its influence on the adsorption of total soluble protein and alpha-glucosidase within various zones of the expanded bed. The retention of the biomass present in the homogenate obtained at a lower intensity of disruption was found to be high at the lower end of the column (17% from 13.8 MPa sample compared to 1% from 41.4 MPa sample). This interaction of the particulate material with the adsorbent was found to reduce the dynamic binding capacity of the adsorbent for total soluble protein from 3.6 mg/mL adsorbent for 41.4 MPa sample to 3.0 mg/mL adsorbent for 13.8 MPa sample. The adsorption of alpha-glucosidase was found to increase with an increase in the concentration of the enzyme in the feed, which increased with the intensity of disruption. Selective adsorption of 6,732 U alpha-glucosidase per mg of total protein bound, was noticed for the feedstock prepared at a higher disruption intensity at 41.4 MPa compared to adsorption of 1,262 U/mg of total protein bound for that prepared at 13.8 MPa. The selective adsorption of alpha-glucosidase due to its high concentration together with simultaneous high specific activity of the enzyme in the feed indicated the significance of selective release of enzymes during microbial cell disruption for efficient expanded bed adsorption processes.     

Improved flow cytometric analysis of the budding yeast cell cycle

The budding yeast, Saccharomyces cerevisiae has been a remarkably useful model system for the study of eukaryotic cell cycle regulation. Flow cytometric analysis of DNA content in budding yeast has become a standard tool for the analysis of cell cycle progression. However, popular protocols utilizing the DNA binding dye, propidium iodide, suffer from a number of drawbacks that confound accurate analysis by flow cytometry. Here we show the utility of the DNA binding dye, SYTOX Green, in the cell cycle analysis of yeast. Samples analyzed using SYTOX Green exhibited better coefficients of variation, improved linearity between DNA content and fluorescence, and decreased peak drift associated with changes in dye concentration, growth conditions or cell size.     

Effect of Yeast Cell Morphology,Cell Wall Physical Structure and Chemical Composition on Patulin Adsorption

The capability of yeast to adsorb patulin in fruit juice can aid in substantially reducing the patulin toxic effect on human health. This study aimed to investigate the capability of yeast cell morphology and cell wall internal structure and composition to adsorb patulin. To compare different yeast cell morphologies, cell wall internal structure and composition, scanning electron microscope, transmission electron microscope and ion chromatography were used. The results indicated that patulin adsorption capability of yeast was influenced by cell surface areas, volume, and cell wall thickness, as well as 1,3-β-glucan content. Among these factors, cell wall thickness and 1,3-β-glucan content serve significant functions. The investigation revealed that patulin adsorption capability was mainly affected by the three-dimensional network structure of the cell wall composed of 1,3-β-glucan. Finally, patulin adsorption in commercial kiwi fruit juice was investigated, and the results indicated that yeast cells could adsorb patulin from commercial kiwi fruit juice efficiently. This study can potentially simulate in vitro cell walls to enhance patulin adsorption capability and successfully apply to fruit juice industry.     

Expanded bed adsorption of human serum albumin from very dense Saccharomyces cerevesiae suspensions on fluoride-modified zirconia.

The adsorption of proteins from high cell density yeast suspensions on mixed-mode fluoride-modified zirconia (FmZr) particles (38 to 75 microm, surface area of 29 m(2)/g and density of 2.8 g/cm(3)) was investigated using human serum albumin (HSA) added to Saccharomyces cerevesiae as the model expression host. Because of the high density of the porous zirconia particles, HSA (4 mg/mL) can be adsorbed from a 100 g dry cell weight (DCW)/L yeast suspension in a threefold-expanded bed of FmZr. The expanded bed adsorption of any protein from a suspension containing >50 g DCW/L cells has not been previously reported. The FmZr bed expansion characteristics were well represented by the Richardson-Zaki correlation with a particle terminal velocity of 3.1 mm/s and a bed expansion index of 5.4. Expanded bed hydrodynamics were investigated as a function of bed expansion using residence time distribution studies with sodium nitrite as the tracer. The adsorption of HSA on FmZr exhibited features of multicomponent adsorption due to the presence of dimers. The protein binding capacity at 5% breakthrough decreased from 22 mg HSA/mL settled bed void volume for 20 g DCW/L yeast to 15 mg HSA/mL settled bed void volume for 40 g DCW/L yeast and remained unchanged for the higher yeast concentrations (60 to 100 g DCW/L). However, the batch (or equilibrium) binding capacity decreased monotonically as a function of yeast concentration (20 to 100 g DCW/L) and the binding capacity at 100 g DCW/L yeast was fivefold lower compared with that at 20 g DCW/L yeast. The lower batch binding capacity at high cell concentrations resulted from the adsorption of cells at the surface of the particles restricting access of HSA to the intraparticle surface area. Batch (or equilibrium) and column HSA adsorption results indicated that the adsorption of HSA on FmZr occurred at a time scale that may be much faster than that of yeast cells. The zirconia particles were cleaned of adsorbed HSA and yeast with a total of 1500 to 2000 column volumes (over many cycles) of 0. 25 M NaOH, without any significant effect on the chromatographic performance.     

酵母菌Y17吸附Cu2+的影响因素及吸附机理研究

以高效吸附Cu2+的酵母菌Y17为材料, 对其吸附Cu2+过程中的主要影响因素, 包括溶液pH、Cu2+初始浓度、菌体添加量、吸附时间和温度以及吸附机理进行了探讨。结果表明, 对吸附过程影响较大的因素依次为吸附液pH值、Cu2+初始浓度、菌体添加量和吸附时间。正交试验得到最佳吸附条件为溶液pH5.0, 吸附时间40 min, 加菌量5.0 g湿菌/L时, 对初始浓度为8 mmol/L的Cu2+达到最佳吸附率为82.7%。通过对Y17菌体不同处理及解吸实验, 初步确定Y17吸附Cu2+的位点在细胞壁, 细胞壁表面的-NH2, -COOH基团在其吸附过程中起着重要作用。     

Adsorption of metal ions on yeast cells at varied cell concentrations

Adsorption isotherms for adsorption of metal ions on yeast cellswere determined at varied cell concentrations. The mass of adsorbedmetal per unit weight of the cells at an equilibrium metal concentrationwas largely dependent on the cell concentration. An experimentalequation was presented, relating the included parameters. (Received August 19, 1975; )     

酵母菌Y17吸附Cu2+的影响因素及吸附机理研究

以高效吸附Cu2 的酵母菌Y17为材料,对其吸附Cu2 过程中的主要影响因素,包括溶液Ph、Cu2 初始浓度、菌体添加量、吸附时间和温度以及吸附机理进行了探讨.结果表明,对吸附过程影响较大的因素依次为吸附液Ph值、Cu2 初始浓度、菌体添加量和吸附时间.正交试验得到最佳吸附条件为溶液Ph5.0,吸附时间40min,加菌量5.Og湿菌/L时,对初始浓度为8mmol/L的Cu2 达到最佳吸附率为82.7%.通过对Y17菌体不同处理及解吸实验,初步确定Y17吸附Cu2 的位点在细胞壁,细胞壁表面的-NH2,-COOH基团在其吸附过程中起着重要作用.     

Ochratoxin A removal in synthetic and natural grape juices by selected oenological Saccharomyces strains

AIMS: To assess, for the first time the efficiency in removing ochratoxin A (OTA) from laboratory medium [yeast peptone glucose (YPG)], synthetic grape juice medium (SGM) and natural grape juice by viable and dead (heat and acid-treated) oenological Saccharomyces strains (five S. cerevisiae and one S. bayanus) compared with a commercial yeast walls additive. METHODS AND RESULTS: Levels of OTA during its interaction with six oenological Saccharomyces strains (five S. cerevisiae and one S. bayanus) or with a commercial yeast walls additive in YPG medium, in SGM or in natural grape juices was assessed by HPLC after appropriate extraction methods. A significant decrease of OTA levels in YPG medium and SGM was observed for many of the growing strains reaching a maximum of 45%, but no degradation products were detected. With both heat and acid pretreated yeasts, OTA removal was enhanced, indicating that adsorption, not catabolism, is the mechanism to reduce OTA concentrations. Adsorption was also improved when the yeast concentration was increased and when the pH of the medium was lower. Approximately 90% of OTA was bound rapidly within 5 min and up to 72 h of incubation with heat-treated cells of either S. cerevisiae or S. bayanus. A comparative study between heat-treated cells (HC) and commercial yeast walls (YW) (used as oenological additive), introduced at two different concentrations (0.2 and 6.7 g l(-1)) in an OTA-contaminated grape juice, showed the highest efficiency by HC to adsorb rapidly within 5 min the total amount of the mycotoxin. CONCLUSIONS: Oenological S. cerevisiae and S. bayanus were able to remove ochatoxin A from synthetic and natural grape juices. This removal was rapid and improved by dead yeasts having more efficiency than commercial yeast walls. SIGNIFICANCE AND IMPACT OF THE STUDY: The efficiency of heat-treated yeasts to remove OTA gives a new hope for grape juice and must decontamination avoiding negative impacts on human health.     

Osmotic mass transfer in the yeast Saccharomyces cerevisiae.

This paper reviews the passive mechanisms involved in the response of a yeast to changes in medium concentration and osmotic pressure. The results presented here were collected in our laboratory during the last decade and are experimentally based on the measurement of cell volume variations in response to changes in the medium composition. In the presence of isoosmotic concentration gradients of solutes between intracellular and extracellular media, mass transfers were found to be governed by the diffusion rate of the solutes through the cell membrane and were achieved within a few seconds. In the presence of osmotic gradients, mass transfers mainly consisting in a water flow were found to be rate limited by the mixing systems used to generate a change in the medium osmotic pressure. The use of ultra-rapid mixing systems allowed us to show that yeast cells respond to osmotic upshifts within a few milliseconds and to determine a very high hydraulic permeability for yeast membrane (Lp>6.10(-11) m x sec)-1) x Pa(-1)). This value suggested that yeast membrane may contain facilitators for water transfers between intra and extracellular media, i.e. aquaporins. Cell volume variation in response to osmotic gradients was only observed for osmotic gradients that exceeded the cell turgor pressure and the maximum cell volume decrease, observed during an hyperosmotic stress, corresponded to 60% of the initial yeast volume. These results showed that yeast membrane is highly permeable to water and that an important fraction of the intracellular content was rapidly transferred between intracellular and extracellular media in order to restore water balance after hyperosmotic stresses. Mechanisms implied in cell death resulting from these stresses are then discussed.     

Improved Flow Cytometric Analysis of the Budding Yeast Cell Cycle

The budding yeast, Saccharomyces cerevisiae has been a remarkably useful model system for the study of eukaryotic cell cycle regulation. Flow cytometric analysis of DNA content in budding yeast has become a standard tool for the analysis of cell cycle progression. However, popular protocols utilizing the DNA binding dye, propidium iodide, suffer from a number of drawbacks that confound accurate analysis by flow cytometry. Here we show the utility of the DNA binding dye, SYTOX Green, in the cell cycle analysis of yeast. Samples analyzed using SYTOX Green exhibited better coefficients of variation, improved linearity between DNA content and fluorescence, and decreased peak drift associated with changes in dye concentration, growth conditions or cell size.

Key Words:

Flow cytometry, Cell cycle, Saccharomyces cerevisiae, SYTOX Green, Propidium iodide     

Detection of Active Yeast Cells (Saccharomyces cerevisiae) in Frozen Dough Sections

A new method based on fluorescence microscopy was developed to detect active yeast cells in cryosections of wheat dough. The sections were stained with 4′,6-diamidino-2-phenylindole (DAPI) and counterstained with Evans blue. The active yeast cells in the sections appeared brilliant yellow and were readily distinguished from the red dough matrix. The dead cells allowed penetration of the Evans blue through the cell membrane, which interfered with the DAPI staining and caused the dead cells to blend into the red environment. The number of active yeast cells in fermenting dough sections containing different proportions of living and dead yeast cells correlated well with the gas-forming capability of the yeast in the dough but not with the results of the conventional plate count method. The new method allows the study of yeast activity not only during the different stages of frozen dough processing but also during the fermentation of doughs.     

Non-electrostatic interactions between cultured Saccharomyces cerevisiae yeast cells and adsorbent beads in expanded bed adsorption: Influence of cell wall properties

The present work focused on the importance of performing EBA experiments under real process conditions. To this end, a high ionic strength medium was used. The influence of cultivating Saccharomyces cerevisiae yeast cells on their subsequent adsorption on a Q HyperZ anion-exchanger was investigated. Two types of industrial yeast cells were used. Once cultured, both types of cells presented similar hydrophilic surface properties and identical adsorption profiles on the anion-exchanger. This was significantly different from the results obtained in previous work on the same yeast cells, just rehydrated in saline buffer, a biological model widely used in the literature. It was postulated that unavoidable “sticky” compounds, initially present in the culture medium or formed during the drying process, were strongly adsorbed on the cell wall and could not be completely removed during the successive washings of the rehydrated cell suspension before use. This could dramatically alter the yeast surface properties and modify the biomass/adsorbent interactions and the bed hydrodynamics, thus demonstrating the necessity to work with yeast coming from fresh cultures. Using biologically active yeast cells allowed to really elucidating the main physico-chemical mechanisms involved in cell adsorption by focusing on the role of the non-electrostatic interactions. Two laboratory mutant yeast strains in which the protein-related cell wall biogenesis was affected, were tested. No significant differences were observed between hydrophilic and hydrophobic yeast cells: the bed remained stable at its initial value and there was a low adsorption ratio in a narrow range between 10% and 17%. This clearly demonstrated that non-electrostatic interactions play a minor role on the affinity of yeast to anion-exchanger adsorbents.     

Metabolic flux-based modeling of mAb production during batch and fed-batch operations

This paper proposes mathematical models that predict the physiology, growth behavior and productivity of hybridoma cells in both batch and fed-batch systems. Murine hybridoma 130-8F producing anti-F-glycoprotein monoclonal antibody was employed as a model system. A systematic approach based on metabolic flux analysis (MFA) was utilized to yield a dynamic model for predicting the concentration of significant metabolites over time. Correlation analysis was performed to formulate a Biomass Model for predicting cell concentration and viability as a function of the extracellular metabolite concentrations. The coefficients of the model equation were estimated by employing the Metropolis–Hastings algorithm. The Metabolites Model was combined with the Biomass Model to get an Integrated Model capable of predicting concentration values for substrates, extracellular metabolites, and viable and dead cell concentration by utilizing only starting concentrations as input. The prediction accuracy of the model was tested by using experimental data.     

酵母发酵蔗渣半纤维素水解物生产木糖酶

采用二次正交旋转组合设计研究了蔗渣半纤维素水解过程中硫酸浓度与液 固比对木糖收率的影响。回归分析表明 ,这两个因素与木糖的收率之间存在显著的回归关系。通过回归方程优化水解条件 ,当硫酸浓度 2 .4g L ,液 固 =6 .2 ,在蒸汽压力 2 .5× 10 4Pa的条件下水解 2 .5h ,10 0g蔗渣可水解生成木糖约 2 4g。大孔树脂吸附层析处理蔗渣半纤维素水解物 ,能有效地减少其中的酵母生长抑制物含量 ,显著改善水解物的发酵性能。用大孔树脂在pH 2条件下处理过的蔗渣半纤维素水解物作基质 ,含木糖 2 0 0g L ,产木糖醇酵母菌株CandidatropicalisAS2 .1776发酵 110h耗完基质中的木糖 ,生成木糖醇 12 7g L ,产物转化率 0 .6 4(木糖醇g 木糖g) ,产物生成速率 1.15g L·h .     

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)).