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To investigate the effects of lactate on cell growth and antibody production, a new method of maintaining the lactate concentration constant in a fed-batch culture is described. When the pH was initially adjusted by sodium hydroxide, the specific growth rate decreased and specific death rate increased with an increase of lactate concentration. To investigate whether the inhibition was due to the lactate concentration itself or to the osmotic pressure, the effect of the osmotic pressure adjusted by sodium chloride was compared with that of sodium lactate. When the osmotic pressure was adjusted to same condition as that of sodium lactate using sodium chloride, the specific growth data showed the same degree of growth inhibition. It was thus evident that the inhibition to cell growth was mainly due to osmotic pressure while lactate production from glucose was found to be inhibited by the lactate itself compared with sodium chloride. The specific antibody production rate had a maximum value within a certain range of lactate concentration. Moreover, specific antibody production rate had a unified relationship with the kinetic parameter mu, in spite of the different causes of inhibition by lithium lactate and sodium lactate. A certain "trade-off" relationship between growth and antibody production existed at higher growth rates.

Fluorescent-labeled invertase, a hyperglycosylated mannoprotein from Saccharomyces cerevisiae, was found to bind to Lactococcus lactis IL1403 at acidic pH. Proteins on the cell wall of the bacterium affinity-purified using invertase as a ligand were identified to be heat shock proteins such as DnaK and GroEL and glycolytic enzymes such as pyruvate kinase and glyceraldehyde-3-phosphate dehydrogenase. DnaK bound to both the bacterium and yeast at pH 4 and aggregated them at above 0.1 mg/ml, whereas no significant difference between the circular dichroism spectra of DnaK at pH 4 and 7 was observed. These results indicate that the cytosolic proteins, including DnaK displayed on the cell wall, cause the lactic acid bacterium to adhere to the yeast.

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A new isolated bacterial strain A-04 capable of producing high content of polyhydroxyalkanoates (PHAs) was morphologically and taxonomically identified based on biochemical tests and 16S rRNA gene analysis. The isolate is a member of the genus Ralstonia and close to Ralstonia eutropha. Hence, this study has led to the finding of a new and unexplored R. eutropha strain A-04 capable of producing PHAs with reasonable yield. The kinetic study of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] production by the R. eutropha strain A-04 was examined using butyric acid and gamma-hydroxybutyric acid as carbon sources. Effects of substrate ratio and mole ratio of carbon to nitrogen (C/N) on kinetic parameters were investigated in shake flask fed-batch cultivation. When C/N was 200, that is, nitrogen deficient condition, the specific production rate of 3-hydroxybutyrate (3HB) showed the highest value, whereas when C/N was in the range between 4 and 20, the maximum specific production rate of 4-hydroxybutyrate (4HB) was obtained. Thus, the synthesis of 3HB was growth-limited production under nitrogen-deficient condition, whereas the synthesis of 4HB was growth-associated production under nitrogen-sufficient condition. The mole fraction of 4HB units increased proportionally as the ratio of gamma-hydroxybutyric acid in the feed medium increased at any value of C/N ratio. Based on these kinetic studies, a simple strategy to improve P(3HB-co-4HB) production in shake flask fed-batch cultivation was investigated using C/N and substrate feeding ratio as manipulating variable, and was successfully proved by the experiments.