Eiji Nagamori

Lipase production in two-step fed-batch culture of organic solvent-tolerant Pseudomonas aeruginosa LST-03.

Efficient lipase production by two-step fed-batch culture of an organic solvent-tolerant bacterium, Pseudomonas aeruginosa LST-03, was investigated. When FB synthetic medium was used in flask culture, no lipase activity was detected, whereas lipase was produced at 2.3 I.U./ml in C2 complex medium. However, lipase production was induced in FB medium when a fatty acid was added to the culture broth in the stationary phase. Among fatty acids tested, long chain saturated fatty acids, such as C18 (stearic acid) and C20 (arachidic acid), were found to function as effective inducers for the production of lipase, giving an activity level almost the same as that obtained in C2 medium in flask culture. Two-step lipase production, comprised of a growth phase in fed-batch mode and a production phase in which lipase was induced by the addition of 5% (v/v) stearic acid, was carried out in a jar-fermentor. In the growth phase, the maximum cell concentration at 16 h was only 20 in terms of the optical density at 660 nm (OD660), and a low level of lipase production (8 I.U./ml) was obtained after 167 h. This was considered to be due to the exhaustion of several medium components brought about by the use of an unsuitable medium or feeding solution. After analyzing the contents of the compounds in the culture broth by inductively coupled plasma spectrometry for metal ions and HPLC for anions, a modified FB medium was designed. When this modified FB medium was used in two-step fed-batch culture, the maximum cell concentration reached an OD660 of 55 (30.2 g-dry cells/l) at 16.5 h, and lipase was produced at 96 I.U./ml after 35 h, which is approximately 40 times higher than the production level obtained in flask culture using C2 medium.

Enhanced anthocyanin production from grape callus in an air-lift type bioreactor using a viscous additive-supplemented medium.

An N-medium containing carboxymethyl cellulose (CMC) was applied to an air-lift type bioreactor culture of grape (Vitis vinifera cv. Bailey alicant A.) callus, and anthocyanin production was investigated. Grape callus grew well at an air flow rate of 80 ml/min and anthocyanin production was significantly increased in the N-medium, reaching 17 mg/l after 7 d of culture. The anthocyanin content of the N-medium was about two times higher than that of the conventional medium without CMC. The effect of air flow rate was also investigated within the range from 40 to 160 ml/min. A twofold increase in anthocyanin content was obtained at all the air flow rates tested in the N-medium. The distribution of grape callus size obtained after 7 d of the bioreactor culture was investigated. The average callus size was 490 mum which was 1.6 times larger than that obtained in the conventional medium. It was found that large calli with a relatively high anthocyanin pigment content were formed in the bioreactor culture using the N-medium. The fluid dynamics in the bioreactor was also investigated at three points (top, middle and bottom) in the bioreactor by laser doppler velocimetry. The average axial velocity of the circulated medium was 0.4 times lower than that of the conventional medium while their average radial velocities were almost the same (zero). The standard deviation of radial velocity fluctuation in the N-medium was also 0.4 times less than that in the conventional medium. These results suggest that turbulent flow occurred in the bioreactor culture using the conventional medium and the degree of turbulent flow decreased significantly when 0.8% CMC was added to the medium to prepare the N-medium. A change of the flow pattern is considered to be the cause of the decrease in hydrodynamic stress, resulting in enhanced pigment production due to the enlargement of the callus.

Enhancement of anthocyanin production from grape (Vitis vinifera) callus in a viscous additive-supplemented medium

N-medium, which is the medium supplemented with carboxymethyl cellulose (CMC) as a viscous additive, was applied to the anthocyanin production from grape callus, Vitis vinifera cv. Bailey alicant A. Although the cell growth decreased with increasing CMC content in the medium, the anthocyanin content per packed cell volume (PCV) increased significantly. When N-B5-medium (0.8%), B5-medium containing 0.8% CMC, was used, approximately three times higher level of anthocyanin production was obtained than that from the control culture without CMC addition. In the repeated batch culture using N-B5-medium (0.8%), the productivity was found to continue at a high level during 30 batch cultures, while no anthocyanin productivity was obtained after 16th batch in the control culture. When the completely white callus after 16th batch culture was transferred to N-B5-medium (0.8%) and cultured repeatedly, anthocyanin productivity was completely recovered after 6th batch culture in N-B5-medium. In contrast, when calli obtained from N-B5-medium (0.8%) was cultured in conventional medium, the productivity was gradually decreased and became almost 0 after 8th repeated batches. Size distribution of calli obtained from the culture in N-B5-medium (0.8%) was also investigated. After repeated batch culture in N-B5-medium (0.8%), the size of calli was shifted to large size and the fraction of calli with less than 0.2 mm decreased remarkably. All calli in the fraction of more than 0.6 mm in size contained pigmented purple cells, while in the fraction of less than 0.2 mm in size the ratio of calli containing pigmented purple cells was less than 50%. From these results, it was confirmed that the enhanced and prolonged production of anthocyanin was due to the shift of size distribution to larger size and it was suggested that the shift of cell size was caused by lowering the damage to grape callus by hydrodynamic stress via increase of medium viscosity.

Enhanced and prolonged production of plantlets regenerated from carrot callus in a viscous additive-supplemented medium

To reduce the hydrodynamic stress in plant cell culture and enhance the production of regenerated plantlets, a liquid medium containing a viscous additive was newly designed and plantlet production from embryogenic carrot callus cultivated in the medium was examined. Na-alginate or carboxymethyl cellulose (CMC) was used as the viscous additive. The viscosity of the medium increased with increasing additive content and the number of regenerated plantlets also increased. When carrot calli were cultivated in the medium containing 0.4% CMC, designated as N medium (viscosity, 3 mPa.s), the maximum enhancement of plantlet regeneration, approximately 2.5 times higher than that in the control medium, was obtained. Enlargement of callus size observed in N medium is considered to be the main reason for the enhanced plantlet regeneration. Regeneration enhancement was sufficiently induced after calli were cultivated once in N medium, but this regeneration ability rapidly disappeared after once cultivation in the conventional medium. In repeated batch culture using N medium, plantlet production continued at a high level for 18 batches (250 d) with no significant decrease, while in the control culture without CMC the number of plantlets produced dropped to almost zero by the sixth batch (84 d).

Release of embryogenic carrot cells with high regeneration potency from immobilized alginate beads

For the mass-production of regenerated carrot plantlets, embryogenic carrot callus immobilized in calcium alginate gel beads was cultivated in a growth medium and the regeneration frequency of cells released from alginate gel beads was compared with that in a suspension culture. Cells released in the immobilized culture were regenerated at a frequency which was about 1.5 times higher than that obtained in the suspension culture. When CaCl2 was added to the growth medium at 5 mM, repeated batch culture for plantlet production continued for 245 d with no significant decrease in the productivity (1.6 x 10(5) plantlets/l-medium/d).