Takuo Yano

Prediction of the concentration of several constituents in a mouse-mouse hybridoma culture by near infrared spectroscopy

Abstract Near infrared spectroscopy (NIR) was employed to predict the concentrations of the nutrients, glucose and glutamine, and the products, lactic acid and ammonia, in the culture broth of a mouse-mouse hybridoma. A supernatant of the culture broth was supplied to a near infrared spectrophotometer and the absorbance at wavelengths between 1,100 and 2,500 nm was measured. The correlations between the concentrations obtained by conventional methods and those obtained by NIR were examined by multiple regression analysis. NIR values predicted by 5-wavelength regression were in good agreement with those obtained by the usual methods. The multiple correlation coefficient was over 0.98 for all four substances. The procedure involved in NIR is simple, and the operation time required to predict the concentrations is only 5 min. The results suggest that NIR is a useful method for the control of animal cell cultures.

Optimum Substrate Feed Rate in Fed-Batch Culture with the DO-Stat Method

Abstract In a fed-batch culture employing the DO-stat method, a rapid increase of dissolved oxygen concentration due to a lack of substrate (the DO signal) is used as an indicator for substrate feeding. The amount of substrate to be fed in response to the appearance of the DO signal is a very important factor for obtaining an optimal fed-batch culture. To select the optimum amount of substrate to be fed at the DO signal, a calculative procedure based on the growth yield, and the relationship between the specific growth rate and substrate concentration is proposed. This procedure is demonstrated in fed-batch cultures of Protaminobacter ruber (a methanol-utilizing bacterium) and Candida brassicae (an ethanol-utilizing yeast). The optimum feed rates calculated with the procedure, 2.5 ml (methanol/ l /signal) for P. ruber and 5 ml (ethanol/ l /signal) for C. brassicae , both gave good agreement with the cultivation results.

Extraction of volatile fatty acids from diluted aqueous solution using a supported liquid membrane

Experimental and analytical studies on the extraction of volatile fatty acids (VFAs) from an aqueous solution using a supported liquid membrane (SLM) were carried out. Teflon and 20% (w/w) tri-n-octyl phosphine oxide (TOPO) in kerosene were used as the supporting membrane and liquid, respectively. The extraction rate of VFAs transferred from the source across the SLM to the sink was measured. It was observed that only undissociated forms of VFAs can penetrate into the SLM and that the complex formation between TOPO and VFA (1/1 molar ratio) inside the membrane enhanced the transfer rate of VFA across the membrane. This phenomenon was explained by mathematical models based on mass transfer and the chemical reactions occurring inside the membrane, suggesting that diffusion of the VFA-TOPO complex inside the membrane may be the rate-limiting step in this experiment.

Biomethanation of H2 and CO2 by Methanobacterium thermoautotrophicum in membrane and ceramic bioreactors

Abstract Direct conversion of gaseous H 2 and CO 2 to CH 4 was achieved with Methanobacterium thermoautotrophicum ΔH (DSM 1053) cells fixed either on a cellulose acetate membrane or inside a porous silica-alumina ceramic support. In a membrane bioreactor with cellulose acetate (5 μmo), methane production rate increased in proportion to the contact area between the gases and the methanogen cells, giving a methane production rate of 0.75 ml CH 4 /cm 2 contact area/h. The initial fixed-cell mass of 0.2 mg dry cell/cm 2 of contact area increased to 1 mg/cm 2 after 12 h of cultivation (steady state). In the ceramic bioreactor (cylindrical, 30 mmo × 70; av. pore size 100 μ, and porosity 79.7%), the methane production rate at steady state was 6 l CH 4 / l ceramic/ l . The methanogen cells grew homogeneously inside the ceramic up to 7 cm depth, and the cell density ranged from 20 to 30 mg dry cell/cm 3 ceramic.

Fed-batch culture with a modified DO-stat method

Abstract To support a high growth rate of microorganism in fed-batch culture with high cell density, a modified DO-stat method was developed. In this method, an exponential substrate feed was coupled with the usual DO-stat method, i.e. , a fixed amount of substrate per DO signal was exponentially fed to the culture based on the estimation of the substrate consumption rate and thereafter the feed was stopped in order to prevent the oversupply of substrate until an abrupt increase in the concentration of dissolved oxygen (DO) in the broth appeared. After that, the feed was started again and this cyclic operation was repeated throughout the cultivation. This method was applied to the fed-batch cultivation of ethanol utilizing yeast, Candida brassicae . At high cell densities (> 10 g/ l ), this modified method was more effective than the usual one in keeping a higher growth rate.

Effect of Dissolved Oxygen Concentration on Growth of Mouse-Mouse Hybridoma

The effects of the dissolved oxygen concentration (DO) in the culture broth on the cell growth, consumption rates of nutritional elements such as glucose and amino acids, and production of anti-IgE antibody, lactic acid and ammonia were studied. Four batch cultures were carried out under the different DO conditions, 75, 50, 20 (corresponded to air saturation, ca.7 ppm) and 6%. With the increase of DO, maximum values of viable cell number and antibody produced decreased, but the amount of ammonia produced was almost constant. The specific growth rate was almost constant below 50% of DO, but at 75% of DO decreased remarkably. The specific production rates of inhibitory compounds, ammonia and lactic acid were minimum at 6% of DO. Cell yield from glucose or glutamine was maximum at 6% of DO, and decreased with the increase of DO. In conclusion, DO level at 6% seemed to be favorable for the effective antibody production by the hybridoma cells.

Measurement of cell density in cultures of aggregative organisms by continuous-dilution-photometric-assay

Abstract A cell density photometric measurement system developed previously was applied to cultures of the aggregative organisms Favolus arcularius and Streptomyces griseus. The system consisted of a photometer with a flow cell, two tubing pumps and a magnetic circulating pump. The culture broth was introduced from a culture flask to the measurement system by a tubing pump, and diluted with water fed by another tubing pump. The diluted broth was circulated by the circulating pump for several minutes to break up the cell aggregates into small pieces. The cell suspension was circulated through the flow cell and diluted with water continuously. The cell density of the culture broth was calculated based on the optical density, sampled volume of the culture broth, water flow rate for dilution, and dilution time, and then converted to dry cell mass concentration by a predetermined calibration curve. The effects of the operating conditions on calculation of the cell density were studied to select the optimum condition. The values of cell densities measured by this method gave good agreement with the values obtained by the drying method over a wide range of cell densities from 5 to 45 g dry cells/l.

Selection of sulfur source for methane production by Methanobacterium thermoautotrophicum

Abstract Sulfur sources capable of replacing sulfide were surveyed for biomethanation from H 2 and CO 2 by thermoautotrophic methanogen, Methanobacterium thermoautotrophicum . Among sulfur containing compounds tested, l -cysteine, thiosulfate and coenzyme M gave poor growth when added as sulfur sources, whereas simultaneous addition of two sulfur sources, l -cysteine+thiosulfate, l -cysteine+ l -methionine or l -cysteine+coenzyme M stimulated the growth. In a pressure-controlled fermentor system developed to obtain stoichiometry between input and output gases, the ratio of H 2 and CO 2 consumption to CH 4 production was almost stoichiometric, and when l -cysteine and thiosulfate or l -methionine were used in place of sulfide (control) similar growth patterns were observed. In a culture with continuous supply of substrates gases (1.3 vvm) and sulfur sources of 1 mM l -cysteine+2 mM thiosulfate, specific growth rate and specific methane production rate were 0.35 h − and 3.24 l g −1 h −1 , respectively, compared to 0.22 h −1 and 5.76 l g − h −1 with Na 2 S.

A Simple Automatic Control System of pH and DO

A simple and inexpensive system to control pH and dissolved oxygen concentration (DO) in the culture broth was developed. To control pH and DO, N2 or CO2 and N2 or O2 were supplied to the head space in a 1L jar-fermentor, respectively. All gas flows were regulated by the ON-OFF mode of each solenoid valve fitted on the gas lines. The effects of the operating conditions such as gas flow rates on the distribution of pH and DO values measured at a certain time interval were examined in the cultivation of a mouse-mouse hybridoma cell. For pH control, over 90% of the measured values was found between the set values, and gas flow rates did not affect the pH control. On the DO control, over 95% of the DO value measured was found between two set values though the under- and over-shoots of the measured value were observed at a higher flow rate of N2 gas. From the performance, the developed system was enough to be applied for animal cell culture.

Extraction of Volatile Fatty Acids from Spent Medium with a Supported Liquid Membrane

Experimental and analytical studies on extraction of volatile fatty acids (VFAs) from aqueous solution using a supported liquid membrane (SLM) were carried out. Teflon soaked with 20% (w/w) of tri-n- octyl phosphine oxide (TOPO) in kerosene was used as a supporting membrane. The extraction rate of VFAs transferred from acid source across the SLM to the sink was measured. It was observed that only undissociated form of VFAs can penetrate into the SLM and the complex formation between TOPO and VFA (1/1 molar ratio) inside the membrane enhanced a flux of VFA across the membrane. This phenomenon was analyzed by the mathematical models for transfer of permeate across the SLM by carrier-facilitating mechanism. In this extraction system, diffusion of the VFA-TOPO complex inside the membrane was most probably the rate-limiting step for the transfer of VFAs.