Kenichi Higashiyama

Production of arachidonic acid byMortierella fungi

The growing interest in the application of arachidonic acid (ARA) in various fields of health and dietary requirements has elicited much attention on the industrial production of ARA-containing oil by the cultivation ofMortierella fungi. For the industrial production of ARA, various studies, such as isolation of a high-potential strain and optimization of culture conditions, have been conducted. Studies including the investigation of morphology are important because ARA is accumulated in the mycelia, and thus cultivation with high biomass concentration is essential for obtaining a high ARA yield. Combining the results derived from various studies, a high ARA yield was attained in an industrial fermentor. These ARA production techniques are applicable to the production of other polyunsaturated fatty acids (PUFAs), and will contribute to the improvement of fermentation technology especially in the field of fungal cultivation.

Effects of dissolved oxygen on the morphology of an arachidonic acid production by Mortierella alpina 1S-4.

Arachidonic acid (AA) production by Mortierella alpina 1S-4 was investigated using a 50-L fermentor. In order to optimize the dissolved oxygen (DO) concentration and to investigate the effect of DO on morphology, cultivation was carried out under constant DO at various levels in the range of 3-50 ppm. To maintain a DO concentration above 7 ppm, two methods, i.e., the oxygen-enrichment (OE) method (experimental range, 25-90% oxygen gas supplied) and the pressurization (PR) method (experimental range, 180-380 kPa headspace pressure), were used. As a result, the optimum DO concentration range was found to be 10-15 ppm. In this optimum DO concentration range, the AA yield was enhanced about 1.6-fold compared to that obtained at 7 ppm DO, and there was no difference in the AA productivity between the OE and PR methods. When the DO concentration was maintained at 20-50 ppm using the OE method, the morphology changed from filaments to pellets, and the AA yield decreased drastically because of stress due to the limited mass transfer through the pellet wall. When the DO concentration was maintained at 15-20 ppm using the PR method, the morphology did not change, and the AA yield decreased gradually.

Effect of consumed carbon to nitrogen ratio of mycelial morphology and arachidonic acid production in cultures of Mortierella alpina.

The influence of the consumed carbon to nitrogen (C/N) ratio on arachidonic acid (AA) production and mycelial morphology was investigated in cultures of Mortierella alpina using shake flasks and a fermentor. The consumed C/N ratio was varied from 5 to 32 under the condition that the total initial amount of carbon and nitrogen sources was 50 g/l. Cellular yield increased markedly at C/N ratios below 7; carbon utilization was switched from cellular growth to lipid biosynthesis in the C/N ratio range of 7-15; lipid biosynthesis was most active when the C/N ratio was in the range of 15-32. However, for C/N ratios higher than 15, the mycelial concentration decreased due to nitrogen limitation but the lipid yield still increased. In the presence of excess nitrogen, the biomass concentration depended on the amount of the nitrogen source, but the AA yield was inversely related to this. On the other hand, in the presence of excess carbon, the fatty acid concentration increased with carbon source concentration but the AA concentration remained constant. From the viewpoint of AA production, the optimum C/N ratio was in the range of 15 to 20 with a balance between the amounts of carbon and nitrogen sources. When an enriched medium was used at a fixed C/N ratio of 20, the cellular and AA concentrations were shown to be proportional to the total concentrations of carbon and nitrogen sources in both flasks and the fermentor. The whole pellet size and width of pellet annular regions did not change with increasing C/N ratio for C/N ratios below 20 in the flask cultures. However, when the C/N ratio was higher than 20, these sizes increased in proportion to the C/N ratio.

The enhancement of specific antibody production rate in glucose- and glutamine-controlled fed-batch culture.

The concentration effects of certain amino acids (Asp, Ile, Leu, Lys, Met, Val, Phe and Gln which were highly consumed during cultivation), and glucose on cell growth and antibody productivity were investigated using dish culture. From these experiments, it was found that only glutamine enrichment enhanced the specific antibody production rate. The other amino acids described above did not affect either the specific growth rate or specific antibody production rate. Thus we investigated the quantitative effects of glutamine concentration in the range of 0.4∼33.3 mmol·1−1 on kinetic parameters in fed-batch culture which kept both glucose and glutamine concentration constant. As a result the specific growth rate decreased with increase in glutamine concentration in the range larger than 20 mmol·1−1. The specific antibody production rate had a maximum value at about 25 mmol·1−1 glutamine concentration.

Image analysis of morphological change during arachidonic acid production by Mortierella alpina 1S-4.

The changes in mycelial morphology during arachidonic acid (AA) production by Mortierella alpina 1S-4 were investigated using an image analysis system. Cultivation was performed in a 10-kl fermentor, and the culture broth was separated into two fractions by sieving (0.5 mm aperture size): the filament fraction (F-fraction, <0.5 mm), and the pellet fraction (P-fraction, >0.5 mm). The effect of the mycelial morphology in each fraction on AA production was analyzed. As a result, a product distribution in the culture broth wherein the AA content in the mycelia of the P-fraction was observed to be higher than that in the mycelia of the F-fraction throughout the cultivation. Morphological analysis of the P-fraction revealed that the hairy pellets became smooth because the mycelia on the pellet surface were shaved off; some pellets were broken and reduced in size. The shaved-off mycelia from the hairy pellets surface moved into the F-fraction and aggregated there. From the above findings, it was likely that the low AA content in the F-fraction was due to mycelial damage during the cultivation. In addition, the morphology of the hairy pellets was found to contribute to an increase in the viscosity of culture broth.

Morphological diversity ofMortierella alpina: Effect of consumed carbon to nitrogen ratio in flask culture

The influence of the consumed carbon to nitrogen (C/N) ratio on mycelial morphology was investigated in cultures ofMortierella alpina using shake flasks. The consumed C/N ratio was varied from 5 to 32 under the condition that the total initial amount of the carbon and nitrogen sources was 50 g/L. The whole mycelia and filamentous mycelia exhibited no relationship with the consumed C/N ratio below a consumed C/N ratio of 20 in the presence of either excess carbon or excess nitrogen. However, when the consumed C/N ratio increased higher than 20, the mycelial sizes increased in proportion to the consumed C/N ratio. However, the area ratio of filamentous mycelia to total mycelia was found to be independent of the consumed C/N ratio, and remained constant at 0.82. In the case of a fixed consumed C/N ratio of 20, the whole mycelia and filamentous mycelia increased in proportion to the degree of the medium strength, yet the area ratio of filamentous mycelia to total mycelia remained unchanged at 0.76. Accordingly, these results show that fungal morphology and mycelial size are both affected by the ratio of carbon to nitrogen. The findings of the current study will be helpful in obtaining the efficient production of useful bioproducts from fungal cultures.

Dielectric analysis for estimation of oil content in the mycelia of Mortierella alpina

The dielectric behavior of the filamentous fungi Mortierella alpina SAM2104 and 1S-4, which produce polyunsaturated fatty acid enriched oil in the mycelia, was investigated. During the cultivation carried out in a 10-kL fermentor for 12-15 days, the relative permittivity and conductivity of the broth were measured in the frequency range of 100 kHz to 30 MHz. The dielectric parameters, i.e., the amplitude of dielectric relaxation (Deltaepsilon) and the characteristic frequency (f(c)), were obtained by fitting the Cole-Cole equation to the observed dielectric relaxation, and the conductivity of the medium (kappa(a)) was also measured. The value of Deltaepsilon gradually increased from the second day through the end of cultivation, suggesting that volume fraction of the cell increased with oil accumulation. The conductivity of the cytoplasm (kappa(i)) was calculated from the experimental values of f(c) and kappa(a), using a theoretical equation based on an ellipsoidal cell model. As a result, good correlation between the calculated kappa(i) and the oil content was obtained. These findings indicate that dielectric analysis enables us to estimate the oil content in the mycelia of oleaginous fungi and also provides a useful tool for monitoring cell growth and for controlling the cultivation process.

Analysis of morphological relationship between micro- and macromorphology of Mortierella species using a flow-through chamber coupled with image analysis

ABSTRACT. Using a flow‐through chamber coupled with image analysis, the morphological parameters of 11 Mortierella species were quantified, and the relationship between micro‐ and macromorphology was investigated. On potato‐dextrose‐agar plates, 5 species formed rose petal‐like colonies, 3 formed large round colonies, and 3 formed donut‐like colonies. By observing micromorphology in a flow‐through chamber, fungi were divided into 3 groups, classified according to morphological parameters: (i) a group with a high branch formation rate (qb: tip/μm/h) and a low tip extension rate (qtip: μm/tip/h); (ii) a group with a low branch formation rate and a high tip extension rate; and (iii) a group intermediate between the former and the latter groups. In suspension culture, group (i) fungi formed a hyphal bundle with a pulpy pellet‐like morphology and a pellet core. In contrast, group (ii) fungi showed an aggregation of hyphae without the pellet core. In a narrow‐specific hyphal growth rate (μl) range (0.35–0.45 h−1), a higher branch formation rate led to increased hyphal branching, resulting in the formation of a hyphal bundle with a pulpy pellet‐like morphology and a pellet core. When the branch formation rate was lower than 2 × 10−3 tips/μm/h, the mycelia formed less branched but longer hypha. Our study surmises that a micromorphology consisting of a high hyphal growth rate (0.4 h−1), low tip extension rate (20 tips/μm/h), and high branch formation rate (8 × 10−3 tips/μm/h) forms the suitable macromorphology for arachidonic acid production.

The Effects of Glutamine and Glucose Concentration on Hybridoma Cell Growth and Antibody Productivity

Recently the serum-free culture techniques have been developed and many hybridoma cell lines are able to grow in serum-free medium. The major nutrients in serum-free culture are sugar and amino acids which are important factors for both cell growth and antibody production. In this study, we report on the effect of these concentrated substrates, glucose and amino acids, which are much consumed during cultivation. We investigated quantitatively the glutamine enrichment effect on the antibody production using fed-batch operation which could control glucose and glutamine concentration at a constant value, and the effect of glucose concentration on the cell yield.

The Effects of Glutamine Concentration on Growth and Monoclonal Antibody Production in Fed-Batch Operation

The effects of concentrated amino acids and glucose in the medium on the cell growth and antibody productivity were investigated using dish culture. From these experiments, it was found that only the enriched glutamine enhanced the specific antibody production rate. Hence, we investigated the quantitative effects of glutamine concentration in the range of 0.4∼33.3 mmol·l−1 on kinetic parameters by the fed-batch culture which could keep glucose and glutamine concentration constant at the same time. The specific growth rate decreased with the increase of glutamine concentration in the range larger than 20 mmol·l−1 and the specific antibody production rate had a maximum value at about 25 mmol·l−1 of glutamine concentration.