Osamu Hiruta

Application of Maxblend Fermentor® for microbial processes

Abstract Application of the Maxblend® impeller (which was originally developed for mixing high viscous liquids during chemical reactions) to a bioreactor (Maxblend Fermentor® [MBF]) was investigated. In comparison with turbine impeller fermentor (TBN), the MBF showed higher mixing capacity for high viscous liquids and consequently, the mixing time (θ m) was less than 50% of that observed with the TBN. Furthermore, by combining the Maxblend® impeller with a spiral sparger, the kLa was much higher than that of the TBN with a ring sparger. During the fermentative production of hyaluronic acid (a biopolymer) in TBN, there was a nonhomogeneous pH distribution inside the fermentor when the viscosity of the broth was high. Consequently, the pH in some parts of the fermentor was lower than the optimum pH for hyaluronic acid production. On the other hand, when MBF with spiral sparger was used, the pH distribution inside the fermentor was homogeneous throughout the cultivation, resulting to about 20% increase in the hyaluronic acid productivity compared with a TBN. In the case of γ-linolenic acid (GLA) production by Mortierella ramanniana, although the power consumption for liquid agitation was lower in the MBF equipped with a spiral sparger than in the TBN, small hard pellets were formed in former and this resulted in a higher GLA content of the lipid. Furthermore, in contrast with the pellets of various sizes formed in the TBN, the sizes of the pellets formed in the MBF were uniform. This was because the shear stress distribution was more uniform inside the MBF than in the TBN under the same agitation conditions.

Optimization and scale-up of γ-linolenic acid production by Mortierella ramanniana MM 15-1, a high γ-linolenic acid producing mutant

Abstract Optimum conditions for cultivation of Mortierella ramanniana mutant MM 15-1 were investigated with an aim of producing a lipid which contains high γ-linolenic acid (GLA). The optimum initial glucose concentration and pH control level were 300 g/ l and 4.0, respectively. Although both pellet and filamentous forms of this mutant were observed during the cultivation, the pellets accumulated lipid with higher GLA content than the filamentous forms. The effects of culture conditions on pellet formation were therefore investigated in a 30- l jar fermentor. The results showed that with an inoculum spore concentration of 5.0 × 10 3 /ml and an agitation speed of 800 rpm, 0.15–0.5 mm pellets were formed and consequently, the GLA content of the lipid increased to a very high value of 18.3%. Furthermore, when this process was scaled up to a 10-kl fermentor, using the impeller tip velocity as the scale-up parameter, both the pellet formation and the lipid production as well as the GLA content of the lipid were consistent with those obtained in the 30- l jar fermentor.

γ-Linolenic acid production by a low temperature-resistant mutant of Mortierella ramanniana

As a result of breeding experiments using low temperature growth as an index, Mortierella ramanniana mutant MM 15-1, which produces lipids with a high γ-linolenic acid (GLA) content, was obtained. The results of cultivation experiments in Erlenmeyer flasks showed that although the total lipid produced by the MM15-1 mutant was about 14% lower than that of the parent strain (M. ramanniana IFO 8187), the GLA content of the lipid was 16.5%, which represents about a 2-fold increase over that of the parent strain. In particular, there was a significant increase in the GLA proportion of the phospholipid fraction in the MM15-1 mutant. Furthermore, the results of the lipid composition analysis after phospholipid fractionation by TLC showed that there was a significant increase in the GLA proportions in the phosphatidylethanolamine (PE) and phosphatidylinositol (PI) of the MM 15-1 mutant. When the MM 15-1 mutant was cultivated in a 600-l fermentor, it produced lipid with 18.3% GLA. This was a 2-fold higher than the value obtained with the parent strain under the same cultivation conditions.

Scale-up of bialaphos production

Abstract Scale-up of bialaphos production from a 3 l jar fermentor to a 300 kl fermentor was investigated. When the scale-up was based on power consumption (for agitation) per unit volume of the reactor, bialaphos production in the 300 kl fermentor was remarkably lower than that obtained with the 3 l jar fermentor. The reason for the unsuccessful scale-up was investigated by considering the major differences between the 3 l jar and the 300 kl fermentor. It was found that variation in the shear stress had no significant effect on bialaphos production. A special pressure-proof fermentor with pressure control system was used to study the effect of liquid pressure gradient in the 300 kl fermentor. When the cells in the special fermentor were subjected to cyclic variation in pressure (between 1,013 and 1,994 HPa) as though they are circulated between the top and the bottom of a 10 m liquid level in the 300 kl fermentor, bialaphos production was as low as that obtained in the 300 kl fermentor. The optimum DO concentration for bialaphos production in the 3 l fermentor was found to be 0.5 ppm and above this concentration, an inverse relationship between the DO concentration and bialaphos production was observed. The inhibition of bialaphos production at high pressure was therefore attributed to the increase in the DO concentration at high liquid pressures. When the scale-up was based on the DO concentration and the DO at the middle of the 300 kl fermentor was controlled at 0.5 ppm, bialaphos production increased to 85 % of that obtained with the 3 l jar fermentor. By taking the DO concentration gradient in the 300 kl fermentor into consideration, the DO at the bottom of the fermentor was maintained at 0.5 ppm. This resulted in a successful scale-up wherein bialaphos production increased to 96% of the maximum production in the 3 l jar fermentor.

Estimation of microbial cell concentration in suspension culture by the osmotic pressure measurement of culture broth

A method for estimating microbial cell concentration in suspension cultures even under heterogeneous conditions was developed on the basis of changes in osmotic pressure of the medium. During batch cultivation of Saccharomyces cerevisiae and Candida brassicae, there was a linear relationship between increase in cell concentrations (ΔX) and the difference between osmotic pressure change in the broth (ΔPc) and the osmotic pressure increase due to product accumulation (ΔPp) regardless of the product (ethanol) concentration in the broth. A linear relationship between (ΔX) and (ΔPc — ΔPp) was also observed when medium containing solid substrate (wheat germ) was used. An enzymatic method for separating cells from the solids was developed and cell concentrations in broths containing solid substrates could be measured accurately. During the batch production of bialaphos (a herbicide) by Streptomyces hygroscopicus using a medium containing solid substrates, the cell concentration could also be estimated by the developed methods.

Effect of oxygen partial pressure on bialaphos synthesis, sugar metabolism and the activity of tricarboxylic acid cycle enzymes in submerged culture of Streptomyces hygroscopicus☆

Abstract Bialaphos [a herbicide, 2-amino-4-(hydroxy)(methyl)-phospinoylbutyryl-alanyl-alanine] produced by Streptomyces hygroscopicus increased significantly as the oxygen supply decreased during both growth and production phases of batch culture. Under low oxygen partial pressure, there were decreases in sugar consumption by the cells, cell concentration and the activity of tricarboxylic acid cycle enzymes in the cells. On the other hand, the activity of glyoxylic acid cycle enzymes in the cells increased. These phenomena were more prominent in high producing than in low producing strains. From these results, it is suggested that in high bialaphos producing strains, low oxygen partial pressure suppresses the activity of tricarboxylic acid enzymes so that both acetyl-CoA and pyruvate (the substrates for bialaphos synthesis) are used mainly for secondary metabolism. This results in a high production of bialaphos.

Relationship between sugar consumption and tricarboxylic acid cycle enzyme activity in a high bialaphos-producing strain☆

Abstract The biosynthesis of bialaphos [a herbicide, 2-amino-4(hydroxy)(methyl)-phosphinoylbutyryl-alanylalanine] produced by Streptomyces hygroscopicus , proceeds through the degradation of glucose to phosphoenol pyruvate, the formation of a CP bond, acetic addition by acetyl-CoA and alanine addition. Therefore, bialaphos formation is considered to be closely related to glucose metabolism. Based on this hypothesis, sugar consumption and the activities of the tricarboxylic acid cycle enzymes were examined using a highly productive strain and a strain of the lower productivity. It was clear that the highly productive strain has a lower sugar consumption rate and lower yield of cells, compared with the lower productivity strain. The activities of the tricarboxylic acid cycle enzymes of the highly productive strain were lower than those of the lower productivity strain, while the activities of the glyoxylic acid cycle enzymes of the highly productive strain were higher. From these findings, it is suggested that the highly productive strain suppresses the flow from acetyl-CoA and pyruvate (as substrates of bialaphos) to the tricarboxylic acid cycle, and efficiently directs these substrates to the secondary metabolism by activation of the glyoxylic acid cycle, resulting in a high rate of bialaphos production in this strain.

Accumulation of bialaphos intermediates under high oxygen partial pressure and their conversion to bialaphos

Abstract In bialaphos (a herbicide) fermentation by Streptomyces hygroscopicus , production of bialaphos is greatly reduced when the partial pressure of oxygen is increased. In order to determine the inhibitory effect of high oxygen partial pressure, the culture filtrate was analyzed revealing that large amounts of two possible biosynthetic intermediates of bialaphos were accumulated. These two compounds were identified to be C-P compounds having an amino group, one as a demethyl derivative of bialaphos (MP-102) and the other a demethyl derivative of AMPB [dealaninated bialaphos] (MP-101). It was also demonstrated that these demethyl derivatives were converted to bialaphos when they were added to the culture under low oxygen partial pressure, whereas the conversion was suppressed under high oxygen partial pressure. As a conclusion, methylation at the final step of the biosynthetic process of bialaphos did not proceed under high oxygen partial pressure, resulting in a low bialaphos concentration due to the accumulation of the demethyl derivatives.

Enzymatic degradation of poly(hydroxyalkanoate) by Corynebacterium aquaticum IM-1 isolated from activated sludge

Abstract Poly(4-hydroxybutyric acid)-degrading bacteria were isolated from activated sludge. One strain was selected, which was identified as Corynebacterium aquaticum. The strain C. aquaticum IM-1 excreted an extracellular poly(hydroxyalkanoate) [PHA]depolymerase and grew on 4-hydroxybutyric acid as the sole carbon source. The PHA depolymerase was purified from the cultured broth medium containing 4-hydroxybutyric acid as the sole carbon source by ultrafiltration, gel filtration and hydrophobic column chromatography. The molecular weight of the depolymerase was determined as approximately 33000 Da by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The optimum activity of the depolymerase on the degradation of poly(4-hydroxybutyrate) (P(4HB)) was observed at pH 6.5 and 40 °C. Lipase activity of the depolymerase was not detected, and N-terminal sequences of the depolymerase were not applicable to the well-known enzymes. The enzymatic degradation of polyesters was studied by the erosion rate of solution-cast films of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) and poly(ω-hydroxyalkanoate) of different chain lengths (C3–C6). The water-soluble products of poly(3-hydroxybutyrateco-97mol%4-hydroxybutyrate) film were revealed by 1H NMR and FAB-MS analysis, and the main product was the dimer of 4-hydroxybutyric acid.