Crossflow filtration of yeast broth cultivated in molasses

A broth of yeast cells cultivated in molasses was crossfiltered with a thin-channel module. The permeation flux gradually decreased at a constant cell concentration. The flux was much lower than that obtained for yeast broth cultivated in yeast extract, polypeptone, and dextrose (YPD) medium during the filtration. The flux did not depend on the membrane pore size (0.45 to 5 mum). The steady-state flux was one-twentieth that calculated for a cake filtration mode from the amount of cake per unit filtration area and the specific resistance of the cake measured in a dead-end filtration apparatus. The lower flux was due to small particles (most of which were less than 1 mum in diameter) in the molasses. The mehanism of crossflow filtration of broths of yeast cells cultivated in molasses was clarified by analysis of the change in flux with time and observations with scanning electron microscopy. At the initial stage of crossflow filtration the yeast cells and particles from the molasses were deposited on the membrane to form the molasses were deposited on the membrane to form a cake in a similar way to dead-end filtration. After the deposition of cells onto the membrane ceased, the fine particles from molasses formed a thin layer, which had higher resistance than the cake formed next to the membrane. The backwashing method was effective to increase the flux. The flux increased low when the pore size was 0.45 to 0.08 mum, but using larger pores of 3 to 5 mum it returned almost to the bases line. (c) 1994 John Wiley & Sons, Inc.     

Microfiltration of recombinant yeast cells using a rotating disk dynamic filtration system

To develop a highly efficient cell harvest step under time constraint, a novel rotating disk dynamic filtration system was studied on the laboratory scale (0.147-ft.(2) nylon membrane) for concentrating recombinant yeast cells containing an intracellular product. The existing cross-flow microfiltration method yielded pseudo-steady state flux values below 25 LMH (L/m(2). h) even at low membrane loadings (10 L/ft.(2)). By creating high shear rates (up to 120,000(-1)) on the membrane surface using a rotating solid disk, this dynamic filter has demonstrated dramatically improved performance, presumably due to minimal cake buildup and reduced membrane fouling. Among the many factors investigated, disk rotating speed, which determines shear rates and flow patterns, was found to be the most important adjustable parameter. Our experimental results have shown that the flux increases with disk rotating speed, increases with transmembrane pressure at higher cell concentrations, and can be sustained at high levels under constant flux mode. At a certain membrane loading level, there was a critical speed below which it behaved similarly to a flat sheet system with equivalent shear. Average flux greater than 200 LMH has been demonstrated at 37-L/ft.(2) loading at maximum speed to complete sixfold concentration and 15-volume diafiltration for less than 100 min. An order of magnitude improvement over the crossflow microfiltration control was projected for large scale production. This superior performance, however, would be achieved at the expense of additional power input and heat dissipation, especially when cell concentration reaches above 80 g dry cell weight (DCW)/L. Although a positive linear relationship between power input and dynamic flux at a certain concentration factor has been established, high cell density associated with high viscosity impacted adversely on effective average shear rates and, eventually, severe membrane fouling, rather than cake formation, would limit the performance of this novel system. (c) 1995 John Wiley & Sons, Inc.     

Crossflow filtration of baker's yeast with periodical stopping of permeation flow and bubbling

The periodical stopping of permeation flow was applied to increase the permeation flux in crossflow filtration of commercially available baker's yeast cell suspension. The permeation flux after 3 h filtration in the crossflow filtration increased to 8 x 10(-5) m(3) /m(2) s (290 L/m(2) h) from 2 x 10(-5) m(3)/m(2) s (72 L/m(2) h) by applying the periodical stopping of permeation. Introduction of air bubbles during the stopping period of permeation further increased the flux.(c) John Wiley & Sons, Inc.     

Real-time fuzzy-knowledge-based control of Baker's yeast production

A real-time fuzzy-knowledge-based system for fault diagnosis and control of bioprocesses was constructed using the object-oriented programming environment Small-talk/V Mac. The basic system was implemented in a Macintosh Quadra 900 computer and built to function connected on line to the process computer. Fuzzy logic was employed in handling uncertainties both in the knowledge and in measurements. The fuzzy sets defined for the process variables could be changed on-line according to process dynamics. Process knowledge was implemented in a graphical two-level hierachical knowledge base. In on-line process control the system first recognizes the current process phase on the basis of top-level rules in the knowledge-base. Then, according to the results of process diagnosis based on measurement data, the appropriate control strategy is subsequently inferred making use of the lower level rules describing the process during the phase in question. (c) 1995 John Wiley & Sons, Inc.     

Gas phase biotransformation reaction catalyzed by baker's yeast

The gas phase continuous production of acetaldehyde from ethanol and hexanol from hexanal using dried baker's yeast was studied as an alternative approach to conventional processes. The effects of water activity, activity of substrates, and amount of yeast on the performance of the continuous bioreactor were investigated. The extent of yeast hydration and ethanol activity are the most important factors affecting yeast activity and stability.     

Reductive biotransformations of organic compounds by saccharomyces cerevisiae: study of oxidoreductases involved using electrophoretic zymograms

The role of oxidoreductases in reduction of carbonyl compounds was investigated by application of zymogram techniques. Eight bands were observed using ethanol with nicotinamide adenine dinucleotide (NAD) as coenzyme. Bands observed with lactic acid and (R)-(-)-phenyl-1,2-ethanediol with nicotinamide adenine dinucleotide phosphate (NADP) had similar R(m) values. 2-Hydroxyvalerate and malate manifested bands having similar R(m) values and were active with both NAD and NADP. Based on their structural similarity and identical R(m) values, oxidation of 1,4-cyclooctanediol (band #2) and cis-1,5-cyclooctanediol may be due to a common enzyme. The PAGE-zymogram technique may be used on a preparative scale to facilitate purification and full characterization on the observed stained bands.     

New technical concept for alternating tangential flow filtration in biotechnological cell separation processes

Robust cell retention devices are key to successful cell culture perfusion. Currently, tangential flow filtration (TFF) and alternating tangential flow filtration (ATF) are most commonly used for this purpose. TFF, however, suffers from poor fouling mitigation, which leads to high filtration resistance and product retention, and ATF suffers from long residence times and cell accumulation. In this work, we propose a filtration system for alternating tangential flow filtration, which takes full advantage of the fouling mitigation effects of alternating flow and reduces cell accumulation. We have tested this novel setup in direct comparison with the XCell ATF® as well as TFF with a model feed comprising yeast cells and bovine serum albumin as protein at harsh permeate to feed flow conditions. We found that by avoiding the dead-end design of a diaphragm pump, the proposed filtration system exhibited a reduced filtration resistance by approximately 20% to 30% (depending on feed rate and permeate flow rate). A further improvement of the novel setup was reached by optimization of phase durations and flow control, which resulted in a fourfold extension of process duration until hollow fiber flow channel blockage occurred. Thus, the proposed concept appears to be superior to current cell retention devices in perfusion technology.     

Simulation of L/A control in fed-batch culture of baker's yeast

L/A controllers have extended their use from continuous to fed-batch fermentation where the control is applied from the start of an initial batch phase. As opposed to proportional integral derivative (PID) controllers where even a startup procedure is recommended prior to fed-batch, the L/A controller is not upset by an early connection. It is easily retuned continuously by means of ethanol measurements and can cope with a large range of output conditions. The performance of an L/A algorithm, which uses biomass concentration as the controlled variable, is assessed through simulation. The self-contained algorithm is relatively simple with no greater intrinsic complexity than modern PID stand alone controllers.     

Pulsatile flow filtration of yeast cell debris: influence of preincubation on performance

The recovery of proteins from yeast cell debris suspension was investigated using a vortex mixing technique based on the combination of oscillatory flow and a baffled flat-sheet microfiltration system. For this system, increased protein transmission was obtained through the use of low transmembrane pressures and the preincubation of the yeast cell debris feed suspension at 30 degrees C. Furthermore, a plateau in the flux-time curve was observed. In the absence of baffles and pulsations, preincubation had a little effect. (c) 1993 Wiley & Sons, Inc.     

Reduction of α-Azidopropiophenone by Immobilized Baker's Yeast

Baker's yeast immobilized on montmorillonite K10 and chrysotile reduced α-azidopropiophenone to the corresponding azidoalcohols syn and anti with good chemical yield and enantiomeric excess.     

Design and evaluation of control strategies for high cell density fermentations

A methodology for the design and evalution of bioprocess control strategies is presented. The strategies manage nutrient supply with demand and vary with the metabolic condition and phase of fermentation operation. Six carbon source addition strategies are based on different combinations of available measurements; they are described and evaluated under different operating conditions for yeast cultivation. It is concluded that a single control strategy is not the most appropriate under all possible operating conditions. An oxygen uptake rate-based control strategy performs better with a mean respiratory quotient (RQ) value less than 1.1 during an oxygen limitation than an ethanol control strategy which had a mean RQ of 14. The designed strategies and an approach of applying the strategy that best matches fermentation conditions consistently enables achievement of high cell densities 78.7 g DCW/L and yields 0.50 g DCW/g glucose as the mean values for three fermentations.     

Integration of enzyme, strain and reaction engineering to overcome limitations of baker's yeast in the asymmetric reduction of alpha-keto esters

We report on the development of a whole-cell biocatalytic system based on the popular host Saccharomyces cerevisiae that shows programmable performance and good atom economy in the reduction of alpha-keto ester substrates. The NADPH-dependent yeast reductase background was suppressed through the combined effects of overexpression of a biosynthetic NADH-active reductase (xylose reductase from Candida tenuis) to the highest possible level and the use of anaerobic reaction conditions in the presence of an ethanol co-substrate where mainly NADH is recycled. The presented multi-level engineering approach leads to significant improvements in product optical purity along with increases in the efficiency of alpha-keto ester reduction and co-substrate yield (molar ratio of formed alpha-hydroxy ester to consumed ethanol). The corresponding alpha-hydroxy esters were obtained in useful yields (>50%) with purities of > or =99.4% enantiomeric excess. The obtained co-substrate yield reached values of greater than 1.0 with acetate as the only by-product formed.     

Comparison of different options for harvest of a therapeutic protein product from high cell density yeast fermentation broth

Recovery of therapeutic protein from high cell density yeast fermentations at commercial scale is a challenging task. In this study, we investigate and compare three different harvest approaches, namely centrifugation followed by depth filtration, centrifugation followed by filter-aid enhanced depth filtration, and microfiltration. This is achieved by presenting a case study involving recovery of a therapeutic protein from Pichia pastoris fermentation broth. The focus of this study is on performance of the depth filtration and the microfiltration steps. The experimental data has been fitted to the conventional models for cake filtration to evaluate specific cake resistance and cake compressibility. In the case of microfiltration, the experimental data agrees well with flux predicted by shear induced diffusion model. It is shown that, under optimal conditions, all three options can deliver the desired product recovery ( >80%), harvest time ( <15 h including sequential concentration/diafiltration step), and clarification ( <6 NTU). However, the three options differ in terms of process development time required, capital cost, consumable cost, ease of scale-ability and process robustness. It is recommended that these be kept under consideration when making a final decision on a harvesting approach.     

Investigation of the parameters affecting the separation of bacterial enzymes from cell debris by tangential flow filtration

The effect of organism, enzyme, method of cell breakage and membrane characteristics on the separation of bacterial enzymes from cell debris by tangential flow filtration has been studied. The effectiveness of separation was assessed by process time, enzyme yield and specific activity, and turbidity of the filtrate. For a particular organism and enzyme, method of cell breakage and membrane characteristics significantly influenced separation performance, though results indicate that it is not yet possible to optimize all aspects of performance simultaneously.     

History,lineage and phenotypic differentiation of sake yeast

ABSTRACT

Sake yeast was first isolated as a single yeast strain, Saccharomyces sake, during the Meiji era. Yeast strains suitable for sake fermentation were subsequently isolated from sake brewers and distributed as Kyokai yeast strains. Sake yeast strains that produce characteristic flavors have been bred in response to various market demands and individual preferences. Interestingly, both genetic and morphological studies have indicated that sake yeast used during the Meiji era differs from new sake yeasts derived from Kyokai Strain No. 7 lineage. Here, we discuss the history of sake yeast breeding, from the discovery of sake yeast to the present day, to highlight the achievements of great Japanese scientists and engineers.     

Increasing yeast secretion of heterologous proteins by regulating expression rates and post-secretory loss

Heterologous protein secretion involves the coupled processes of protein synthesis, protein folding, and secretory trafficking. A more complete understanding of how these processes interrelate could help direct optimization of secretion systems. Here we provide a detailed study regarding the dynamics of heterologous protein secretion from yeast in terms of intracellular protein levels, secreted protein levels, and unfolded protein response (UPR). Three different protein expression induction temperatures (20, 30, and 37 degrees C) were investigated as a means to modulate expression rates and thus cellular responses. Inducing at 20 degrees C yielded the slowest initial secretion rate, but the highest absolute level of product. Correspondingly, the level and the rate of both intracellular protein accumulation and unfolded protein response (UPR) activation were also the lowest at 20 degrees C. In addition, secretion ceased after approximately 22 h at 30 and 37 degrees C, respectively, while it was continuous until nutrient depletion at 20 degrees C. Maxima in secretion levels were observed that were a result of the additive effects of secretion cessation and post-secretory protein loss. The post-secretory loss of protein did not appear to result from solution phase proteolysis or aggregation, but required the presence of yeast cells. Refeeding of both yeast nitrogen base and casamino acids successfully prevented the post-secretory loss of protein at both high (37 degrees C) and low (20 degrees C) temperatures, and further increased secretion levels 1.5-fold at 20 degrees C where the secretory pathway was still functioning. Taken together, these findings suggest that there exists an appropriate balance between protein synthesis, processing and secretion rates required for secretion optimization.     

Production of ethanol from guava pulp by yeast strains

Guava pulp used for ethanol production by three yeast strains contained 10% (w/v) total sugars and was pH 4.1. Ethanol production at the optimum sugar concentration of 10%, at pH 4.1 and 30°C was 1.5%, 3.6% and 3.9% (w/v) by Saccharomyces cerevisiae MTCC 1972, Isolate-1 and Isolate-2, respectively, at 60 h fermentation. Higher sugar concentrations at 15 and 20% were inhibitory for ethanol production by all test cultures. The maximum production of ethanol at optimum natural sugar concentration (10%) of guava pulp, was 5.8% (w/v) at pH 5.0 by Isolate-2 over 36 h fermentation, which was only slightly more than the quantity of ethanol produced by Saccharomyces cerevisiae (5.0%) and Isolate-1 (5.3%) over 36 and 60h fermentation, respectively.     

Stereoselective bioreduction of 1-(5-phenylfuran-2-yl)-ethanones mediated by baker's yeast

Abstract

Baker's yeast mediated reduction of various phenylfuran-2-yl-ethanones has been studied. The influence of the reaction conditions, the type and position of the substituents, as well the presence of various additives on the enantiomeric composition of the products and the reaction yield are discussed. The absolute configuration of the reaction products was established using a retrosynthetic procedure.     

Genetic and fermentation properties of the K2 killer yeast, Saccharomyces cerevisiae T206

Saccharomyces cerevisiae T206 K⁺R⁺, a K₂ killer yeast, was differentiated from other NCYC killer strains of S. cerevisiae on the basis of CHEF-karyotyping and mycoviral RNA separations. Genomic DNA of strain T206 was resolved into 13 chromosome bands, ranging from approximately 0.2 to 2.2 Mb. The resident virus in strain T206 yielded L and M RNA species of approximately 5.1 kb and 2.0 kb, respectively. In micro-scale vinifications, strain T206 showed a lethal effect on a K^-R^- mesophilic wine yeast. Metabolite accumulation and toxin activity were measured over a narrow pH range of 3.2 to 3.5. Contrary to known fermentation trends, the challenged fermentations were neither stuck nor protracted although over 70% of the cell population was killed. Toxin-sensitive cells showed cytosolic efflux.     

Acquisition of thermotolerant yeast Saccharomyces cerevisiae by breeding via stepwise adaptation

A thermotolerant Saccharomyces cerevisiae yeast strain, YK60‐1, was bred from a parental strain, MT8‐1, via stepwise adaptation. YK60‐1 grew at 40°C, a temperature at which MT8‐1 could not grow at all. YK60‐1 exhibited faster growth than MT8‐1 at 30°C. To investigate the mechanisms how MT8‐1 acquired thermotolerance, DNA microarray analysis was performed. The analysis revealed the induction of stress‐responsive genes such as those encoding heat shock proteins and trehalose biosynthetic enzymes in YK60‐1. Furthermore, nontargeting metabolome analysis showed that YK60‐1 accumulated more trehalose, a metabolite that contributes to stress tolerance in yeast, than MT8‐1. In conclusion, S. cerevisiae MT8‐1 acquired thermotolerance by induction of specific stress‐responsive genes and enhanced intracellular trehalose levels. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1116–1123, 2013