Toshiomi Yoshida

Limited feeding of potassium nitrate for intracellular lipid and triglyceride accumulation of Nannochloris sp. UTEX LB1999

Abstract Limited feeding of nitrate during culture of Nannochloris sp. UTEX LB1999 for intracellular lipid and triglyceride accumulation was investigated with the aim of obtaining cells superior for liquefaction into a fuel oil. The intracellular lipid contents and the percentage of triglycerides in the lipids of cells grown in a nitrogen-limited medium (0.9 mM KNO3) were 1.3 times as high as those grown in a modified NORO medium containing 2.0–9.9 mM KNO3. However, the cell concentration was too low for the practical production of fuel oil by high-pressure liquefaction of the cell mass. A single feeding of 0.9 mM nitrate after nitrate depletion during cultivation in a nitrate-limited medium increased the cell concentration to twice that obtained without such feeding, and the lipid content was maintained at a high level. The timing of nitrate feeding, i.e., whether it was given during the log phase (before nitrate depletion), the constant growth phase (just after the depletion), or the stationary phase (after the depletion), had negligible effect on the intracellular lipid content and percentage of triglycerides in the lipids. When 0.9 mM nitrate was intermittently fed ten times during the log phase in addition to the initial nitrate feed (0.9 mM), the cell concentration reached almost the same (2.16 g/l) and the intracellular lipid content and the percentage of triglycerides in the lipids increased from 31.0 to 50.9% and 26.0 to 47.6%, respectively, compared with those of cells cultured in a modified NORO medium containing 9.9 mM KNO3 without additional nitrate feeding.

Construction of xylose-assimilating Saccharomyces cerevisiae

Abstract The xylose reductase gene originating from Pichia stipitis was subcloned on an expression vector with the enolase promoter and terminator from Saccharomyces cerevisiae . The transformants of S. cerevisiae harboring the resultant plasmids produced xylose reductase constitutively at a rate about 3 times higher than P. stipitis , but could not assimilate xylose due to the deficient conversion of xylitol to xylulose. The xylitol dehydrogenase gene was also isolated from the gene library of P. stipitis by plaque hybridization using a probe specific for its N-terminal amino acid sequence. The gene transferred into S. cerevisiae was well expressed. Furthermore, high expressions of the xylose reductase and xylitol dehydrogenase genes in S. cerevisiae were achieved by introducing both genes on the same or coexisting plasmids. The transformants could grow on a medium containing xylose as the sole carbon source, but ethanol production from xylose was less than that by P. stipitis and a significant amount of xylitol was excreted into the culture broth.

The effect of osmolarity on metabolism and morphology in adhesion and suspension chinese hamster ovary cells producing tissue plasminogen activator

The effects of constant osmolarity, between 300 and500 mOsm/kg, on the metabolism of Chinese HamsterOvary (CHO) cells producing tissue plasminogenactivator (tPA) were compared between adhesion andsuspension cultures. In both suspension and adhesionculture, the specific rates of glucose consumption(νG), lactate production (qL), and tPAproduction (qtPA) increased as osmolarityincreased, while these rates decreased when osmolaritywas higher than the respective critical levels. However, specific growth rate (μ) decreased withincrease in osmolarity and this slope grew steeper inthe osmolarity range higher than the critical level. The decrease in μ in the adhesion culture was morerapid than that in the suspension culture. Thecritical osmolarity for adhesion culture (400 mOsm/kg)was lower than that for suspension culture (450 mOsm/kg). These results indicated that the adhesionculture was more sensitive to increase of osmolaritythan the suspension culture, while the specific ratesobtained from the adhesion cultures were in general1.5- to 3-fold higher than those obtained from thesuspension cultures. Cell volume increased asosmolarity increased in both the suspension andadhesion cultures, as reported previously forsuspension culture of hybridoma cells, but there wasno morphological change in the suspension culture. Incontrast, cell height decreased and cell adhesion areamarkedly increased as osmolarity increased in theadhesion culture. This morphological change inadhesion cultures may be one reason for the highersensitivity of adherent cells to the increase ofosmolarity than suspended cells.

High-density cultivation of Perilla frutescens cell suspensions for anthocyanin production: Effects of sucrose concentration and inoculum size☆

High-density cultivation of Perilla frutescens cells for anthocyanin production was carried out in both batch and fed-batch modes in a 500-ml shake flask. In fed-batch cultures, a high cell density of 27.7 g dry cells l−1 and a total anthocyanin production of 3.87 g l−1 by intermittent feeding of all medium components except hormones were obtained. In batch cultures, both initial sucrose concentration and inoculum size showed a conspicuous effect on the kinetics of cell growth, sugar consumption, and secondary metabolite (anthocyanins) production by suspended P. frutescens cells. At an inoculum size of 50 g wet cells l−1, the maximum cell density of 38.3 g dry cells l−1 was obtained after 11 days of cultivation at an initial sucrose concentration of 60 g l−1, the highest pigment production of >5.8 g l−1 was attained after 10 days of cultivation at an initial sucrose concentration of 45 g l−1. These amounts of cell mass and anthocyanin pigments were 3.3 and 24 times higher than those at an initial sucrose concentration of 15 g l−1 and inoculum size of 15 g wet cells l−1, respectively.

Isolation of xylose reductase gene of Pichia stipitis and its expression in Saccharomyces cerevisiae.

A NADPH/NADH-dependent xylose reductase gene was isolated from the xylose-assimilating yeast,Pichia stipitis. DNA sequence analysis showed that the gene consists of 951 bp. The gene introduced inSaccharomyces cerevisiae was transcribed to mRNA, and a considerable amount of enzyme activity was observed constitutively, whereas transcription and translation inP stipitis were inducible.S. cerevisiae carrying the xylose reductase gene could not, however, grow on xylose medium, and could not produce ethanol from xylose. Since xylose uptake and accumulation of xylitol byS. cerevisiae were observed, the conversion of xylitol to xylulose seemed to be limited.

Physiologically motivated strategies for control of the fed-batch cultivation of recombinant Escherichia coli for phenylalanine production

Abstract The efficiency of the fed-batch cultivation of recombinant Escherichia coli AT2471 for phenylalanine production is highly dependent on the distribution of the carbon flow between the main process products — biomass, phenylalanine, acetic acid and carbon dioxide. In order to enhance the process performance, the effects of several factors, namely glucose feeding, tyrosine feeding and oxygen supply, were investigated experimentally. As a result, a set of control strategies was developed, designed to tolerate phenylalanine synthesis at the expense of the remaining products. The DO was controlled to prevent acetic acid excretion due to oxygen limitation. The total amount of tyrosine fed was used to provide an optimal balance between biomass synthesis and that of phenylalanine. Special algorithms for control of the glucose feed rate were applied to eliminate the threat of acetic acid excretion due to overfeeding, and at the same time, to reduce excessive CO 2 evolution caused by unnecessarily severe glucose limitation. The joint application of these strategies resulted in greatly improved efficiency in the phenylalanine production process: the final phenylalanine concentration reached 46 g/ l , the yield was above 17%, and the productivity-0.85 g/ l ·h. In combination, these data exceed the results reported by others, and are much higher than those obtained by use before the implementation of the proposed complex of techniques.

Enhancement of anthocyanin production by Perilla frutescens cells in a stirred bioreactor with internal light irradiation

Abstract Oxygen supply and light irradiation exhibited significant influence on the production of anthocyanin (red pigments) by suspended cultures of Perilla frutescens cells in a 2.6- l aerated and agitated bioreactor with a six-flat-bladed turbine. When the initial volumetric oxygen transfer coefficient ( k L a ) value was below 10 h −1 and light was not irradiated, the anthocyanin production was never over 0.6 g/ l . By modification of a gas sparger, the oxygen supply capability of the bioreactor was remarkably improved, and 1.65 g/ l of anthocyanin was obtained at an enhanced k L a value of 15.4 h −1 . Moreover, it was found that anthocyanin accumulation at a 0.2 vvm aeration rate was higher than that at 0.1 or 0.4 vvm in the modified bioreactor, with the other cultivation conditions kept the same. Light irradiation also significantly increased anthocyanin accumulation in the stirred reactor at a low k L a value, i.e. 9.9 h −1 . However, a combination of irradiation with a higher oxygen supply reduced the production of anthocyanin in the bioreactor.

Computer control of glutamic acid production based on fuzzy clusterization of culture phases

The reaction mechanism in a microorganism is much more complicated than that of an ordinary chemical reactor. The mechanism is fundamentally programmed by the DNA sequences of the organism involved, which can change from their original situation with time. It is difficult to keep all of the characteristics of a microorganism constant for a long period, and the improvement of strains by genetic engineering and/or screening techniques is often tried in order to improve industrial fermentation processes. As a result, models of a real fermentation process usually work only for a limited period, and it is virtually impossible to construct a comprehensive and robust model. Furthermore, only a few kinds of sensors are available for monitoring the culture states in fermentation processes, and we cannot measure the state inside cells directly. Therefore, a deterministic model for the application to the control or the simulation of fermentation processes cannot easily be constructed.

Global and local neural network models in biotechnology: Application to different cultivation processes

Biological processes are non-deterministic systems. In general, models dealing with microbial pathways and microbial physiology are exceedingly complex, and as it is almost impossible to measure intracellular concentrations on-line, they normally have too many uncertain parameters that are difficult to evaluate. It is important, therefore, to generate modeles based on on-line measured variables, as these can be used in process control and on-line optimization of biological systems. Neural networks, which offer a data-driven modeling approach, are well suited for the above purpose, having good generalization and prediction capabilities. Neural networks include, among others, the feedforward-backpropagation and recurrent types. Localized networks such as Radial Basis Function (RBF) networks have found use in on-line process control due to the fact that they require less computational time. Another type of network, Cascade Correlation (CC), has been developed to generate the optimal structure for a particular application. Important factors to be considered in selecting and using neural networks in biotechnology are: proper scaling of data, selection of an appropriate network structure—including suitable choices of input and output variables, and the purpose of the network. In this paper, the above issues and different networks are studied in context with different microbiological systems. Case studies dealing with fuel alcohol production using renewable biomass from agricultural wastes by fermentation with Zymomonas mobilis and recombinant Escherichia coli, and preliminary results for the production of monoclonal antibody using hybridoma cells, are examined. The results of these studies indicate that RBF networks are unsuitable when extrapolation is desired. Among Multi-layer Perceptron (MLP) networks, Recurrent Neural networks and Cascade Correlation networks provide the best prediction capabilities.

Dielectric measurement to monitor the growth and the physiological states of biological cells

Measurement of capacitance, also referred to as dielectric permittivity, is a new method of estimating the concentration of cells, monitoring the growth and detecting the physiological changes during the cultivation of organisms in various bioprocess. Several types of biological cells were studied, namely; Saccharomyces cerevisiae, Escherichia coli, Perilla frutescens (plant cells) and AFP-27 hybridoma cells. Generally, a linear correlation between cell capacitance (C) and other biomass measurement technique such as optical density (OD) and dry weight (DW) was obtained using the different types of cell suspension. Therefore, this method could be used to monitor the growth of the organism during the active growth. It could be conveniently used to make a rapid estimate of the cell concentration such as in plant cell suspension culture. The capacitance sensor could also be designed to be installed and autoclaved in-situ in a bioreactor and used for on-line monitoring of cell growth. On the other hand, distinct deviations in the capacitance value were observed in relation with the growth stage of the organism. This was observed in all the organisms studied but the type of deviation depends on the physiology of the organism. This variation in cell capacitance showed the possibility of using this method as a means to indicate changes in the physiological state of cells during cultivation. This capability would be very useful in designing control strategies that would depend on the physiological states in the bioprocess.