Masahito Taya

Comparison of growth properties of carrot hairy root in various bioreactors

SummaryGrowth properties of carrot hairy root cells in various bioreactors were investigated. A turbine-blade reactor and an immobilized rotating drum reactor were found to be advantageous for the hairy root culture because of a high oxygen transfer coefficient (k in L a). After 30 days of culture, 10 g/l of dry hairy root cells were obtained in both bioreactors and maximum growth rates (Vm) were found to be 0.63 and 0.61 g/l per day for the turbine-blade reactor and immobilized rotating drum reactor, respectively. Specific growth rates (μ) at various cultivation times were observed to be linearly proportional to X/kla for both bioreactor configurations where X is the cell concentration. The estimated specific oxygen uptake rate of 0.34 mmol O2/g dry cells per hour compares fairly well with an experimental value of 0.3.

A general framework for the assessment of extractive fermentations

Abstract A mathematical framework was developed for the assessment of batch, repeated-batch and repeated fed-batch extractive fermentations of acetone and butanol or ethanol. Several useful expressions were derived for determining the noninferior set defined in the two-objective function space, which is concerned with the productivity and the concentration of the metabolic products. The computer simulation based on the experimental data revealed the possibility of attaining a significant improvement for extractive fermentations as compared with conventional fermentations without extraction.

Kinetic expression for human cell growth in a suspension culture system

Abstract The effects of lactate and ammonium concentrations on the growth of HL-60 and RPMI 8226 human cells were kinetically evaluated in a suspension culture system. The specific growth rates of the cells were expressed as the following equation with the terms of the inhibitory effects owing to lactate, ammonium, and glucose. mu;=mu; m S {(K s +S)(1+ P 1 k 1 + P 2 k 2 )+ S 2 k 3 } On the basis of this equation, it was possible to stimulate the culture processes of the human cells.

Production of peroxidase with horseradish hairy root cells in a two step culture system

Abstract Horseradish hairy root cells transformed by a soil bacterium Agrobacterium rhizogenes had peroxidase (POD) activity comparable to that of the original plant root tubers. To enhance POD production by the hairy root culture, various additives to the medium were tested including casein hydrolysates and plant extracts. Polypepton addition had a significant effect on the growth and POD production; at low concentrations (below 1 g/ l ) the growth was stimulated, while at high concentrations (3–10 g/ l ), POD activity in the cells was enhanced in spite of a low growth rate. Therefore, the hairy roots were at first cultured in the medium with 1 g/ l Polypepton, and then 5 g/ l Polypepton was added to enhance intracellular POD activity. POD activity in this two step culture system was 5.4 times higher than that in conventional culture in Polypepton-free medium.

Isolation and characterization of an extremely thermophilic, cellulolytic, anaerobic bacterium

SummaryA cellulolyticm obligately anaerobic, extreme thermophile (strain NA10) was isolated from an alkaline hot spring in Nagano Prefecture, Japan. The microorganism was a non-spore-forming, flagellated rod which had a negative reaction to Gram stain, and occurred singly or in pairs. The growth temperature was between 50° C and 85° C with the optimum at 75° C, and the growth pH was between 6.0 and 9.5 with the optimum at 8.1. The anaerobe characteristically fermented cellulose, and produced acetic acid, H2, CO2 (main products) and lactic acid (minor product). The DNA had a base composition of 37.7 mol% guanine+cytosine content.

Cloning and expression in Escherichia coli of a Thermoanaerobacter cellulolyticus gene coding for heat-stable β-glucanase

SummaryA 4.8 kb HindIII fragment of Thermoanaerobacter cellulolyticus DNA cloned in Escherichia coli was shown to direct the synthesis of β-glucanase. The enzyme produced by the transformant was extremely heat-stable and the optimum temperature for the enzyme reaction was 80°C. The cloned enzyme could hydrolyse carboxymethyl cellulose and lichenan, but could not digest laminarin, xylan and cellobiose. Although T. cellulolyticus secreted cellulase(s) into the medium, most of the cloned enzyme activity was detected only in cytoplasm in the recombinant clone.

Kinetic evaluation and characterization of ceramic honeycomb-monolith bioreactor

Abstract Escherichia coli with γ-galactosidase activity or Saccharomyces cerevisiae was immobilized within a thin κ-carrageenan gel film on a ceramic honeycomb-monolith. The characteristics of the monolith bioreactor were evaluated for reaction systems with and without gas evolution. Under various operational conditions, the Peclet number was found to be nearly constant (Pe=6.6) in both reactions systems. Moreover, the Sherwood number was estimated in terms of Reynolds numbers on superficial liquid and gas velocities. The kinetic analysis based upon the dispersion model and liquid film resistance was done to assess the conversion degree of the substrate in the reactor. In ethanol fermentation accompanied by CO2 gas evolution, as a result, the monolith bioreactor was expected to convert much of the substrate.

Kinetic evaluation and monitoring of methanogen culture based upon fluorometry

SummaryAs the fluorescent coenzyme F420 (factor 420) is present exclusively in methanogenic bacteria, its fluorescence was measured for Methanobacterium thermoautotrophicum ΔH and Methanobrevibacter arboriphilus A2. Cell extracts from acetone-treated methanogenic bacteria showed the highest intensities of fluorescence under excitation (EXW) and emission wavelengths (EMW) of 404 and 470 nm, respectively. No significant fluorescence was observed in the extracts from non-methanogenic microorganisms such as Escherichia coli. On the basis of fluorescence spectra, the fluorescence of the extract was considered to be caused by the coenzyme F420. In both methanogen cultures, the specific fluorescence change rate was nearly equal to the specific growth rate, and the ratio of the intensity to the cell concentration was almost constant during the logarithmic growth phase. It was thus possible to monitor the cultures of the methanogenic bacteria using the intensity of fluorescence as an indicator.

Continuous culture of RPMI 8226 human cells

Abstract Continuous culture of RPMI 8226 human hematopoietic cells was performed. The viable cell number and glucose, lactate and ammonium concentrations became constant within 3–4 days at a constant dilution rate. The viable cell number decreased at low and high dilution rates. The growth and product yields slightly depended on the dilution rate, except for product yield for lactate based on cell number. Growth characteristics of these cells at various dilution rates could be expressed by equations considering the maintenance energy in growth yield. Maximum specific growth rate could be evaluated from the wash-out profile and the known inhibition constants.

Expression of a thermostable cellulase gene from a thermophilic anaerobe in Saccharomyces cerevisiae.

Abstract A cellulase gene from a thermophilic anaerobe was recloned in the yeast Saccharomyces cerevisiae . The maximum level of the gene expression in the recombinant yeast was 4.4 times higher than that in the Escherichia coli transformant harboring the same plasmid. Cellulase activity was observed only within the yeast cells. To compare the enzymatic properties of cellulase produced by the yeast and E. coli transformants, cellulases were purified to homogeneous state by only three purification steps of heat treatment, and cellulose affinity and ion exchange chromatographies. The molecular weights of the enzymes produced by the yeast and E. coli were 3.8 × 10 4 and 4.0 × 10 4 , respectively by SDS-polyacrylamide gel electrophoresis. Neither of the enzymes was glycosylated. Although the molecular weights were slightly different, enzymatic properties and thermostability were almost indistinguishable between the enzymes produced by the yeast and E. coli transformants.