Enoch Y. Park

Production of arachidonic acid by Mortierella fungi

SummaryVarious Mortierella fungi were assayed for their productivity of arachidonic acid (ARA). Only strains belonging to the subgenus Mortierella accumulated detectable amounts of ARA together with dihomo-γ-linolenic acid. None of the strains belonging to the subgenus Micromucor tested accumulated these C-20 fatty acids, although they produced a C-18 fatty acid, γ-linolenic enic acid. A soil isolate, M. alpina 1S-4, was found to grow well in a liquid medium containing glucose and yeast extract as carbon and nitrogen sources, respectively. Addition of several natural oils such as olive and soybean oils to the medium increased the accumulation of ARA. Under optimal culture conditions in a 5-1 bench-scale fermentor, the fungus produced 3.6 g/l of ARA in 7 days. On cultivation for 10 days at 28°C in a 2000-1 fermentor, the same fungus produced 22.5 kg/kl mycelia (dry weight) containing 9.9 kg lipids, in which ARA comprised 31.0% of the total fatty acids. On standing the harvested mycelia for a further 6 days, major mycelial fatty acids (i.e. palmitic acid, oleic acid, linoleic acid, etc.) other than ARA rapidly decomposed and the ARA content of the total fatty acids reached nearly 70%.

Enhancement of ε-Polylysine Production by Streptomyces albulus Strain 410 Using pH Control

The enhancement of epsilon-poly-l-lysine (epsilon-PL) production by Streptomyces albulus strain no. 410 (S410) by means of a pH control strategy was investigated. S140 cells produce epsilon-PL at a high concentration if the culture pH remains at about 4.0; however, if it shifts to higher than 4.0, the accumulated epsilon-PL is depolymerized. We therefore suggest a pH control strategy for cell growth and epsilon-PL production aimed at increasing the amount of epsilon-PL produced. The cultivation was divided into two control phases. In phase I, cell growth was accelerated by maintaining the pH at higher than 5.0; in phase II, epsilon-PL production was increased by maintaining the pH at about 4.0. To avoid an increase in the pH during phase II as a result of glucose depletion, the glucose concentration was kept at around 10 g/l by glucose feeding. This control strategy enhanced the production of epsilon-PL to 48.3 g/l from 5.7 g/l in the case of batch culture.

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.

Efficient production of L-(+)-lactic acid using mycelial cotton-like flocs of Rhizopus oryzae in an air-lift bioreactor.

l‐(+) ‐Lactic acid production was enhanced in a culture of Rhizopus oryzae by induction of a mycelial flocs morphology. By conventional culture the morphology of R. oryzae is that of a pellet‐like cake; however, when mineral support and poly(ethylene oxide) are added to the culture, the morphology of R. oryzae takes on a cotton‐like appearance. The formation of these cotton‐like mycelial flocs was induced by the addition of 5 ppm poly(ethylene oxide) into a 12–14 h culture containing 3 g/L of the mineral support before the formation of the conventional pellet morphology. The cotton‐like flocs were also formed in cultures grown in an air‐lift bioreactor. This morphology allowed effective mass transfer inside the flocs and effective fluidity of culture broth in an air‐lift bioreactor. l‐(+) ‐Lactic acid concentration produced by mycelial flocs in an air‐lift bioreactor, with the support and poly(ethylene oxide), was 104.6 g/L with a yield of 0.87 using 120 g/L of glucose as the substrate; for this culture without both, the concentration was 43.2 g/L. These results demonstrate that cotton‐like mycelial flocs are the optimal morphology for use in the air‐lift bioreactor culture of R. oryzae.

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.

Effect of nitrogen source on mycelial morphology and arachidonic acid production in cultures of mortierella alpina

The effects of nitrogen source on arachidonic acid (AA) production and morphological changes during the culture of Mortierella alpina were investigated using an image analysis system. When yeast extract, gluten meal, or corn steep liquor was used, a circular pellet morphology was obtained. However, when Pharmamedia, fish meal, or soybean meal was used, M. alpina formed radial filamentous mycelia. The radial filamentous area in the case of soybean meal was 75% of the total mycelial area. In a jar fermentor culture, M. alpina morphology varied with the cultivation period: (i) at 0-6 h culture, the inoculated pellet-like mycelia were adapted to the new environment, (ii) at 6 h-1 d culture, filamentous mycelia grew exponentially which yielded a feather-like morphology, (iii) at 1-2 d culture, the filamentous mycelia became disentangled as a result of the mechanical agitation; consequently, the proportion of filamentous mycelia was increased, (iv) at 2-4 d culture, mycelia showed stationary growth, but the AA concentration increased rapidly, and (v) at 4-6 d culture, hyphae grew thick radially with the AA concentration continuing to increase gradually. In the case of the cultures with feather-like morphology obtained using soybean meal, the AA yield was 0.14 g/g dry cell weight, which was two times higher than that in cultures grown using yeast extract. These results suggest that the feather-like morphology of culture of M. alpina is suitable for AA production.

Enhanced cellulase production of the Trichoderma viride mutated by microwave and ultraviolet

Cellulase-producing fungi Trichoderma viride were cultured and fermented on the solid-state wheat bran fermentation medium. The characteristics of its carboxymethyl cellulase (CMCase) in the condition of this solid-state fermentation were evaluated, and the optimum culture time, optimum pH and optimum temperature for CMCase activity of T. viride fermented in this solid state were 60h, 5.0 and 50 degrees C, respectively. Carboxymethyl cellulose sodium (CMC-Na) and Congo red were used to screen the strains that had stronger ability to produce enzymes. After the compound mutagenesis by microwave and ultraviolet, seven mutant strains (M-B1-M-B7) were selected and their CMCase activities were assayed. Five of them (M-B1, M-B2, M-B3, M-B5 and M-B7) had significantly stronger ability to produce enzymes than the normal wild type, and they were also very stable for a long period up to 9 generations to produce cellulase. Molecular studies showed that there were some base mutations in endoglucanase I (EG I) genes of mutants M-B1, M-B2, M-B3 and M-B5, but no change in M-B7, suggesting that some amino mutations in EG I proteins caused by base mutations could lead to enhanced cellulase production.

Multiple co-transfection and co-expression of human β-1,3-N-acetylglucosaminyltransferase with human calreticulin chaperone cDNA in a single step in insect cells

Human β‐1,3‐N‐acetylglucosaminyltransferase 2 (β3GnT2) is indispensable for the conversion of lacto‐N‐triose II into lacto‐N‐tetraose and lacto‐N‐neotetraose. In this paper, we report multiple co‐transfection in a single step using two different human cDNAs in an insect cell, β3GnT2 and calreticulin chaperone respectively. This minimized the time required to isolate stably expressing cell line from 12 weeks to 4 weeks and simplified the isolation technique to a one‐step process. We tried to insert as much cDNA as possible and used various concentrations of two antibiotics, Blasticidin and Geneticin, at 25–1500 μg/ml respectively during co‐transfection for the selection of an efficiently expressing stable cell line with no adverse effects. A stably expressing cell line was isolated which expressed β3GnT2 and chaperone simultaneously, which gave an activity of 10.1 m‐units/ml compared with 6.7 m‐units/ml by a cell only carrying β3GnT2. In this study we correlated the activity of β3GnT2 with the amount of β3GnT2 and human calreticulin cDNA in a stably expressing insect cell line simultaneously expressing calreticulin chaperone. When the amounts of chaperone and β3GnT2 cDNA were in a rough ratio of 1:1, the β3GnT2 activity was at a high level. In order to achieve better expression levels of β3GnT2 with less cost and time, efficient ways have to be devised.

Improved expression of fusion protein using a cysteine‐ protease‐ and chitinase‐deficient Bombyx mori (silkworm) multiple nucleopolyhedrovirus bacmid in silkworm larvae

A bacmid system of BmMNPV [Bombyx mori (silkworm) multiple nucleopolyhedrovirus] with both the cysteine‐protease and chitinase genes deleted (BmMNPV‐CP−‐Chi−) was constructed. The viral protease and chitinase activities in the haemolymph of Bombyx mori larvae infected with this BmMNPV‐CP−‐Chi− bacmid were reduced by 95 and 50% respectively. By using this system, a GFPuv–β3GnT2 (green fluorescent protein excited by UV light–β1,3‐N‐acetylglucosaminyltransferase 2) fusion protein was successfully expressed in silkworm larvae with less protein degradation and without larvae liquefaction; β3GnT2 activity improved 2.8‐fold over that of unmodified bacmid. This BmMNPV‐CP−‐Chi− bacmid system provides rapid protein production in silkworms and can be used for the production of recombinant eukaryotic proteins without proteolytic degradation.

Efficient production of fatty acid methyl ester from waste activated bleaching earth using diesel oil as organic solvent

Fatty acid methyl ester (FAME) production from waste activated bleaching earth (ABE) discarded by the crude oil refining industry was investigated using fossil fuel as a solvent in the esterification of triglycerides. Lipase from Candida cylindracea showed the highest stability in diesel oil. Using diesel oil as a solvent, 3 h was sufficient to obtain a yield of approximately 100% of FAME in the presence of 10% lipase from waste ABE. Kerosene was also a good solvent in the esterification of triglycerides embedded in the waste ABE. Fuel analysis showed that the FAME produced using diesel oil as a solvent complied with the Japanese diesel standard and the 10% residual carbon amount was lower than that of FAME produced using other solvents. Use of diesel oil as solvent in the FAME production from the waste ABE simplified the process, because there was no need to separate the organic solvent from the FAME-solvent mixture. These results demonstrate a promising reutilization method for the production of FAME, for use as a biodiesel, from industrial waste resources containing waste vegetable oils.