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.     

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.     

A unified stoichiometric model for oxidative and oxidoreductive growth of yeasts

Yeasts degrade glucose through different metabolic pathways, where the choice of the pathway is dependent on the nature of the limitation in the various substrates. When oxygen is limiting in addition to glucose, yeasts often grow according to a mixture of oxidative and reductive metabolism. Oxygen may be limiting either by supply or by inherent biological restrictions such as the respiratory bottleneck in Saccharomyces cerevisiae or by both. A unified model incorporating both supply and biological limitations is proposed for the quantitative prediction of growth rates, consumption and production rates, as well as key metabolite concentrations during mixed oxidoreductive metabolism occuring as a result of such oxygen limitations. This simple unstructured model can be applied to different yeast strains while at the same time requiring a minimum number of measured parameters. "Estimators" are utilized in order to predict the presence of supply-side or biological limitations. The values of these estimators also characterize the relative importance of oxidative to total metabolism. Results from the aerobic and oxygen-limited chemostat cultures were used to corroborate the model predictions. During these experiments, the heat released by the yeast cultures was also monitored on-line. The model correctly predicted the overall stoichiometry, steady-state concentrations, and rates including heat dissipation rates measured in the various situations of oxygen limitations. Direct continuous measurements such as heat can be used in conjunction with the unified model for on-line proces control. (c) 1992 John Wiley & Sons, Inc.     

Acetic acid formation in Escherichia coli fermentation

Theoretical analysis of cellulase product inhibition (by cellobiose and glucose) has been performed in terms of the mathematical model for enzymatic cellulose hydrolysis. The analysis showed that even in those cases when consideration of multienzyme cellulase system as one enzyme (cellulase) or two enzymes (cellulase and beta-glucosidase) is valid, double-reciprocal plots, usually used in a product inhibition study, may be nonlinear, and different inhibition patterns (noncompetitive, competitive, or mixed type) may be observed. Inhibition pattern depends on the cellulase binding constant, enzyme concentration, maximum adsorption of the enzyme (cellulose surface area accessible to the enzyme), the range in which substrate concentration is varied, and beta-glucosidase activity. A limitation of cellulase adsorption by cellulose surface area that may occur at high enzyme/substrate ratio is the main reason for nonlinearity of double-reciprocal plots. Also, the results of calculations showed that material balance by substrate, which is usually neglected by researchers studying cellulase product inhibition, must be taken into account in kinetic analysis even in those cases when the enzyme concentration is rather low. (c) 1992 John Wiley & Sons, Inc.     

Yeast calmodulin localizes to sites of cell growth

Summary Intracellular localization of calmodulin was examined in the budding yeast,Saccharomyces cerevisiae. Distribution of calmodulin changes in a characteristic way during the cell cycle. Calmodulin localizes to a patch at the presumptive bud site of unbudded cells. It concentrates at the bud tip in small-budded cells, and later it diffuses throughout the entire bud. At cytokinesis, calmodulin is largely at the neck between the mother and daughter cells. Double staining experiments have shown that the location of some polarized actin dots is coincident with that of calmodulin dots. Polarized localization of actin dots is observed in cells depleted of calmodulin, suggesting that calmodulin is not required for localization of the actin dots. Thecdc24 mutant that has a defect in bud assembly at the restrictive temperature fails to exhibit polarized localization of calmodulin, indicating that theCDC24 gene product is responsible for controlling the polarity of calmodulin.     

Relationship of actin organization to growth in the two forms of the dimorphic yeastCandida tropicalis

Summary Candida tropicalis is a dimorphic yeast capable of growing both as a budding yeast and as filamentous hyphae depending upon the source of the carbon used in the culture medium. The organization of F-actin during growth of the yeast form (Y-form) and the hyphal form (H-form) was visualized by rhodamine-conjugated phalloidin by using a conventional fluorescence microscope as well as a laser scanning confocal fluorescence microscope. In single cells without a bud or non-growing hyphae, actin dots were evenly distributed throughout the cytoplasm. Before the growth of the bud or hypha, the actin dots were concentrated at one site. During bud growth, actin dots were located solely in the bud. They filled the small bud and then filled the apical two-thirds of the cytoplasm of the middlesized bud. During growth of the large bud, actin dots which had filled the apical half of the cytoplasm gradually moved to the tip of the bud. In the formation of the septum, actin dots were arranged in two lines at the conjunction of the bud and the mother cell. During hyphal growth, the majority of actin dots were concentrated at the hyphal apex. A line of clustered spots or a band of actin was observed only at the site where the formation of a new septum was imminent. This spatial and temporal organization of actin in both categories of cells was demonstrated to be closely related to the growth and local deposition of new cell wall material by monitoring the mode of growth with Calcofluor staining. Treatment of both forms of cells with cytochalasin A (CA) confirmed the close relationship between actin and new cell wall deposition. CA treatment revealed lightly stained unlocalized actin which was associated with abnormal cell wall deposition as well as changes in morphology. These results suggest that actin is required for proper growth and proper deposition of cell wall material and also for maintaining the morphology of both forms of cells.Abbrevations FM fluorescence microscopy - EM electron microscopy - rh rhodamine - CA cytochalasin A - CD cytochalasin D - PBS phosphate-buffered saline - DMSO dimethylsulfoxide - GA glutaraldehyde     

The xylanase introns from Cryptococcus albidus are accurately spliced in transgenic tobacco plants

The xylanase gene from Cryptococcus albidus contains seven introns. Genomic and cDNA clones under the control of the CaMV 35S promoter were transferred into tobacco plants using Agrobacterium-mediated cell transformation. The genes were transcribed and the mRNAs were amplified by the polymerase chain reaction using primers on each side of the intron region. About 90% of the amplification products from plants transformed with the genomic clone corresponded to the size of the pre-mRNA (1.2 kb) and 10% represented the spliced product (0.85 kb). The 0.85 kb fragment was cloned and sequenced and the result indicated that the introns from the xylanase gene were accurately spliced by the plant cells.     

Rapid determination of plasmid-carrying yeast cells by using an imaging sensor system

A novel imaging sensor system for the determination of plasmid carrying yeast cells was developed. The sensor system consisted of an Silicon Intensifier Target (SIT) video camera, a fluorescent microscope, and a personal computer system equipped with an image memory board. This system was based on the fact that the membrane integrity of only plasmid-carrying cells is lost following cell growth in 5-fluoro-orotic acid (5-FOA) containing medium, and consequently these target cell can be stained with fluorescent probes and detected. In this study, plasmid-carrying cells were detected and their fraction determined in a mixture of both plasmid-carring and plasmid-free cells. A good correlation was observed between the values determined by this sensor system and the conventional method in the 30%-80% range, and one assay was possible within 4 h. This sensor system could be used for the monitoring of plasmid-carrying fraction in recombinant yeast cells during cultivation.     

Optimal production of glutathione by controlling the specific growth rate of yeast in fed-batch culture

The optimal of the specific growth rate was obtained with simple mathematical model in a yeast fed-batch cultures. The model was based on the mass balance around the fed-batch system and the relationship between the specific growth rate, mu, and the specific production rate of glutathione, rho(G). The optimal profile of mu was calculated as a bang-bang type, That is mu, should start from the maximum value, mu(max) and should be kept at mu(max); then mu should be switched to mu(c), which gives a maximum value of rho(G). It was proven from the maximum principle that switching was needed only once, with the switching time from mu(max) to mu(c) depending on the final required glutathione content. Finally, this ideal profile of mu for the maximum production of glutathione was realized by manipulating the substrates feed rate in the fed-batch culture. Using the extended Kalman filter and a programmed-controller/feedback-compensator (PF) system, mu could be controlled at the optimal profile obtained. As a result, the maximum production of glutathione was accomplished fairly successfully. However, further improvement in the controller performance for mu is desired. The control strategy employed here can be applied to other batch reaction processes.     

Some factors influencing the proportion of periplasmic hepatitisB virus pre-S2 antigen in the recombinant yeastHansenula polymorpha

Summary A central composite design (CCD) was used to evaluate, for the purpose of future process optimization, the influence of pH, yeast extract and ammonium chloride concentrations on the proportion of periplasmic hepatitisB pre-S2 antigen in the recombinant yeastHansenula polymorpha. Each factor was tested at five levels, and a second order polynomial model for the proportion of periplasmic antigen was fitted to the treatment combinations. pH showed the greatest effect: the proportion of periplasmic antigen was greatly increased at the higher pH levels. At the higher pH levels used, the proportion of periplasmic antigen was enhanced by a high concentration of ammonium chloride. Additional experiments have confirmed both the validity of the selected model and the optimal conditions found. A significant correlation was found between the proportion of periplasmic antigen and the total yield of antigen. These results indicated that is should be possible to modulate the distribution of the pre-S2 antigen between the periplasm and the cytoplasm of the yeast.