Mitsuyasu Okabe

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.

Enhanced Production of L(+)-Lactic Acid from Corn Starch in a Culture of Rhizopus oryzae Using an Air-Lift Bioreactor

l(+)-Lactic acid production was investigated using 8 Rhizopus strains and various culture conditions. Rhizopus oryzae NRRL395 showed the highest yield of l(+)-lactic acid among various Rhizopus strains. The optimum production medium contained the following: corn starch, 120 g/l; ammonium sulfate, 1.35 g/l; small amounts of mineral salts (potassium phosphate, magnesium sulfate and zinc sulfate). The data obtained for the flask culture were successfully reproduced using a 3 l air-lift bioreactor under the optimum conditions. The yield (based on initial carbon source concentration) and final concentration of l(+)-lactic acid were 85% and 102 g/l, respectively, in the 3 l air-lift bioreactor. The lactic acid was recovered with a yield (based on lactic acid containing in broth) of 90% using 4 steps, i.e., filtration, active carbon treatment, ultra-filtration and concentration by vacuum evaporation. The lactic acid purified was 100% l(+)-form as judged by HPLC.

Breeding of Aspergillus terreus mutant TN-484 for itaconic acid production with high yield

Abstract A strain, designated TN-484, producing itaconic acid with a high yield was successfully obtained by selection using an itaconic acid concentration-gradient agar plate technique after NTG-treatment of Aspergillus terrerus IFO 6365. The itaconic acid concentration produced by TN-484 was 82 g/l from 160 g/l of glucose in a shaking flask, which is 1.3-times higher than that of the parental strain, and the highest value found in a literature survey. Apart from itaconic acid, by-product organic acids were not detected in the culture filtrate by HPLC analysis. The itaconic acid yields from cells and glucose were 1.8 and 1.2 times those of parental strain, respectively. The itaconic acid concentration was also reproduced in an air-lift bioreactor with a working volume of 2.5 l.

Production of l-lactic acid from corncob

The optimum temperature, initial pH, amount of added enzyme and substrate (corncob) for the hydrolysis of corncob by Acremonium cellulase were 35 degrees C, 4.5, 10 u/g-corncob and 100 g/l, respectively. Under the optimum conditions, more than 55 g/l of reducing sugars were hydrolyzed from 100 g/l of corncob to 34 g/l of glucose and 12 g/l of xylose based on dried corncob. More than 25 g/l of L-lactic acid was produced from this enzymatic hydrolyzate and less than 5 g/l of xylose remained in the 3-l airlift bioreactor. The production of L-lactic acid by simultaneous saccharification and fermentation (SSF) was also carried out in the 3-l airlift bioreactor using Acremonium thermophilus (cellulose-producer) and Rhizopus sp. MK-96-1196 (lactic acid-producer). More than 24 g/l of L-lactic acid was produced from 100 g/l of untreated raw corncob.

L(+)-Lactic Acid Production by Repeated Batch Culture of Rhizopus oryzae in Air-Lift Bioreactor

Abstract l (+)-Lactic acid production using Rhizopus oryzae was investigated by varying the inoculated spore concentration in preculture medium. The spore concentration affected mycelial morphology from dispersed filamentous to pellet form. When the inoculated spore concentration was 2 × 106 spores/ml in flask culture, a small pellet was formed in the culture, which yielded the highest (98.2 g/l) l (+)-lactic acid concentration after 48 h culture; when concentration was 2 × 105 spores/ml, clumped and compacted mycelia were observed, which caused a decrease the l (+)-lactic acid concentration (85.7 g/l) after 48 h culture. Following in air-lift bioreactor culture, the morphology was similar to that in flask culture, and when the inoculated spore concentration was 2 × 106 spores/ml in the seed culture broth, the productivity of l (+)-lactic acid was 1.07 g/l/h after 48 h culture. The pellet morphology made it possible to perform 9 cycles of repeated batch culture over 14 d. During the first 6 cycles, the average productivity was 2.02 g/l/h, which was 1.9 fold higher than that from batch culture.

Optimization and scale-up of L-lactic acid fermentation by mutant strain Rhizopus sp. MK-96-1196 in airlift bioreactors

We determined the optimum culture conditions such as inoculum size, initial starch concentration, pH during the fermentation and aeration rate for L-lactic acid production by Rhizopus sp. MK-96-1196 in a 3-l airlift bioreactor. More than 90 g/l of L-lactic acid was produced from only partially enzymatically hydrolyzed corn starch with a production rate of 2.6 g/l/h and a product yield of 87% based on the starch consumed under the optimum conditions in the 3-l airlift bioreactor. Scale-up from the 3-l to a 100-l airlift bioreactor for L-lactic acid fermentation was carried out using V(s)(cm/s) as a scale-up criterion. The production rates and yields of L-lactic acid in both bioreactors appeared to be fairly well correlated with k(L)a (1/h).

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.

Efficient itaconic acid production from raw corn starch

Itaconic acid production by Aspergillus terreus TN-484 from corn starch as a carbon source was investigated. The production medium consisted only of corn starch pretreated by partial hydrolysis with nitric acid at pH 2 before autoclaving at 121°C for 20 min. Higher than 60 g/l of itaconic acid was produced by A. terreus TN-484 in flask fermentation from medium consisting of 140 g/l of corn starch and no nitrogen source or other ingredients. The yield of itaconic acid based on the amount of corn starch consumed was higher than 50%, and the yield was similar to that of crystal glucose. The yield obtained in flask fermentation was successfully reproduced in a 2.5-l air-lift bioreactor.

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.

Enhancement of L(+)-lactic acid production using mycelial flocs of Rhizopus oryzae

L(+)-Lactic acid production was enhanced in the culture of Rhizopus oryzae using mycelial flocs formed by addition of 3 g/L mineral support and 5 ppm polyethylene oxide. By addition of the mineral support, an electrostatic repulsion between mycelia increased by 3.5-fold compared to that of mycelia, which allowed a dispersed growth of R. oryzae in the early growth phase. In conventional culture the morphology of R. oryzae is that of a pellet-like cake, however, when support and polyethylene 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 polyethylene oxide into a 14 h culture containing the mineral support before the formation of the conventional pellet morphology. The cotton-like flocs were also formed in cultures grown in a fermentor. This morphology allowed effective mass transfer inside the flocs and effective fluidity of culture broth in the reactor. L(+)-Lactic acid concentration produced by mycelial flocs in fermentor, with the support and polyethylene oxide, was 103.6 g/L with the yield of 0.86 using 120 g/L of glucose as the substrate for this cultures without both, the concentration was 65.2 g/L. It demonstrates that cotton-like mycelial flocs are the optimal morphology in the culture of R. oryzae.