Showing papers by "Toshiomi Yoshida published in 2000"

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.

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

Abstract: 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.

The construction of an in vitro three-dimensional hematopoietic microenvironment for mouse bone marrow cells employing porous carriers.

Abstract: Spatial development of mouse bone marrow cellsemploying porous carriers was investigated in order todesign a bioreactor with a three-dimensionalhematopoietic microenvironment. Three types of porouscarriers were used for examining the spatialdevelopment of anchorage-dependent primary stromalcells as feeder cells. Stromal cells were found tospread well at a high density on a polyester nonwovendisc carrier (Fibra cel (FC)) under a scanningelectron microscope, while cells on porous cellulosebeads (Microcube (MC), 500 μm pore diameter)spread at a low density; cells on another type ofcellulose porous beads (CPB, 100 μm pore diameter)were globular. Mouse bone marrow cells wereinoculated to dishes containing three types of porouscarriers which shared more than 30% of the bottomsurface in a dish. The concentration of stromal cellsin the well containing FC was lower than that on theother two carriers. However, the weekly output oftotal hematopoietic cell (suspension cells) increasedbetween day 21 and 28 in the culture using FC while itdecreased monotonously in the cultures by use of theother two carriers. The proportion of progenitorcells (BFU-E, CFU-GM) in the total hematopoietic cellpopulation, after showing an initial decrease,increased after 1 week in the culture using FC whilethe proportion decreased monotonously to zero in thecultures using MC and CPB.

Control of L‐phenylalanine production by dual feeding of glucose and L‐tyrosine

Abstract: Control of L-phenylalanine production by a recombinant of Escherichia coli AT2471 by means of the dual feeding of glucose and L-tyrosine was investigated A novel method was developed for on-line monitoring of the maximum glucose uptake rate (MGUR), in which the length of time required for the consumption of added glucose was measured Accumulation of acetic acid was successfully prevented throughout the whole period of the culture when the glucose concentration was kept below 01 g/L by controlling the glucose feeding on the basis of on-line monitoring of the MGUR and the cell concentration with a laser sensorIn a batch culture with glucose feeding, after L-tyrosine was depleted cell growth and the L-phenylalanine production rate decreased along with decreases in the specific enzyme activities of chorismate mutase-p-prephenate dehydratase (CMP) and 3-deoxy-D-arabinoheputulosonate 7-phosphate synthase (DAHP), which are the key enzymes in the L-phenylalanine synthesis pathway Increasing the L-tyrosine feed rate by an appropriate amount, but not so far as to cause L-tyrosine accumulation in the culture, increased the activities of the enzymes and the specific rates of growth and production while the product yield based on glucose consumption decreasedThe average specific rates of growth, production, and MGUR could be expressed as functions of the specific L-tyrosine consumption rate during both the earlier and later periods of L-tyrosine feeding Estimations of the amount of L-phenylalanine produced, the product yield, and the cost factor by using these functions with several different combinations of two specific L-tyrosine consumption rates for two 10-h periods resulted in a suggested optimum L-tyrosine feeding strategy giving a lower specific L-tyrosine consumption rate in the later period, to suppress cell growth, in comparison to that in the earlier period During L-tyrosine feeding, the three specific rates (growth, production, and MGUR) could be successfully controlled by adjusting the specific L-tyrosine consumption rate to the predicted value The cost factor was lowest in this controlled culture, demonstrating experimentally the effectiveness of the strategy (c) 1996 John Wiley & Sons, Inc

Effect of Acetic Acid on Growth and Ethanol Fermentation of Xylose Fermenting Yeast and Saccharomyces cerevisiae

Abstract: Growth of some xylose fermenting yeasts, Candida shehatae, Pichia stipitis CBS5773, fusant F101 and fusant F198, was completely inhibited in xylose medium added with 0.5% v/v acetic acid which caused the reduction of pH to 4.1. Only one xylose fermenting strain, Pachysolen tannophilus NRRL-Y2460, showed relatively low growth and ethanol fermentation. However, in the medium added with 1.0% v/v acetic acid (pH 3.7) all of these strains were completely inhibited. When the medium was adjusted by hydrochloric acid to pH 4.1 and 3.7, all xylose fermenting strains showed almost the same growth as in the medium without pH adjustment (pH 6.2). In glucose medium added with 0.5% v/v acetic acid, various strains of Saccharomyces cerevisiae, M30, Sc90, N1, G/3, G/5, G/2, TJ3 and SH1089, grew with lower specific growth rate and provided lower maximal cell concentration rate than in medium without adding acetic acid (pH 6.2). All strains, except N1, produced slightly higher maximal ethanol concentration. However, all of them yielded lower ethanol production rate. Among S. cerevisiae, strain B120 was more sensitive to acetic acid than the others since its growth was completely inhibited by 0.5% v/v acetic acid. In glucose medium, 0.5% v/v acetic acid did the same role as in xylose medium to xylose fermenting strains. Hence, the xylose fermenting yeasts revealed higher sensitivity to acetic acid than S. cerevisiae.

Analysis of the ammonia metabolism of rat primary hepatocytes and a human hepatocyte cell line Huh 7.

Abstract: Ammonia metabolism of ratprimary hepatocytes and a human hepatocyte cell line,Huh 7, at different concentrations of glutamine,glucose and ammonia was examined. During theincubation of the primary hepatocyte cells, glutamineand ammonia concentrations decreased, that of ureaincreased, and that of glucose remained the same. Inthe case of Huh 7 cells, glucose was consumed rapidly,the concentration of ammonia increased and that of urearemained the same. The major energy sources amongmedium components were glutamine for the primary cellsand glucose for Huh 7 cells, although the primaryhepatocytes may utilize intracellular glycogen asenergy source. As the glutamine concentration in theincubation medium increased, the specific rates of notonly glutamine consumption, but also ammonia productionby the primary cells and Huh 7 cells increased. Besides, specific urea production rate by the primarycells increased then. Increase of glucoseconcentration had no effect on glutamine and ammoniametabolism by both cells, although it increased glucoseconsumption by Huh 7 cells. The incubation of theprimary cells with higher ammonia concentrationincreased all specific rates of glutamine consumption,ammonia consumption and urea production. An increasein the ammonia concentration to 5 mM changed theammonia metabolism from production to consumption andincreased the specific glucose consumption rate. Consequently, increases in the glutamine and ammoniaconcentrations were revealed to have negative andpositive effects, respectively, on decreasing ammoniaconcentration by both of rat primary hepatocytes andHuh 7 cells.

The Influence of Serum Concentration on tPA Production of CHO Cell

Abstract: The effect of initial serum concentration on cell growth, and the production of tissue plasminogen activator (tPA) is investigated across batch fermentation of a Chinese hamster ovary (CHO) growing in newborn calf serum (NCS) medium. It was found that initial serum concentration influenced on CHO cell growth rate and cell yield. At high-serum content, the uptake rate of glucose decreased although the specific growth rate was higher, indicating a serum component becoming growth limiting. An average glucose uptake rate in 10% (v/v) NCS medium was 13.84 mg/10 6 cells/h as in 1% (v/v) NCS medium was 18.17 mg/10 6 cells/h. It was also found that the tPA or the maximum tPA productions increased with initial serum concentration. At 7.12% NCS, the maximum tPA production gave the highest value (5.55 mg/l). The effect of initial serum concentration on the main product was similar to that of major by-product (e.g. lactate and