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Continuous production of phospholipase D (PLD) in an air-lift reactor system was investigated using growing cells of Streptomyces lydicus D-121 immobilized within cross-linked chitosan beads as porous supports. In a continuous culture with the immobilized growing cells, both the activity and productivity were highest at a dilution rate of 0.330 h −1 which was higher than the maximum specific growth rate of the free cells (0.247 h −1 ). The highest PLD activity was obtained at pH 6.0–6.5. The average activity was 12.7 U/ml during 120–384 h in a continuous culture with immobilized cells, while the productivity of the continuous culture (4.19 × 10 3 U/ l ·h) was about 3.3–3.6-fold higher than that of the batch culture (1.16 × 10 3 U/ l ·h) and the repeated-batch culture in the reactor (1.27 × 10 3 U/ l ·h), and about 3-fold higher than that in the case of continuous culture with free cells (1.36 × 10 3 U/ l ·h).

The phospholipase D (PLD)-encoding gene of Streptomyces antibioticus fused with the pectate lyase B (PelB) signal sequence was expressed in recombinant Escherichia coli under the control of the T7 lac promotor. Most of the PLD activity was detected in the culture supernatant. The N-terminal amino acid sequence of the recombinant PLD was identical to that of the wild-type PLD, suggesting that the processing of the PelB signal peptide occurred correctly and that the PLD produced by E. coli was identical to the wild-type enzyme. The production of PLD was improved by genetic and fermentation techniques. About 3 mg of PLD/ l medium was obtained under optimal conditions, comparable to the amount of PLD produced by S. antibioticus . The results of analysis of the distributions of intracellular marker enzymes suggested that neither cell lysis nor periplasmic disruption was the cause of the secretion of PLD.

Output signals of a commercially available on-line laser turbidimeter exhibit fluctuations due to air and/or CO2 bubbles. A simple data processing algorithm and a personal computer software have been developed to smooth the noisy turbidity data acquired, and to utilize them for the on-line calculations of some kinetic variables involved in batch and fed-batch cultures of uniformly dispersed microorganisms. With this software, about 103 instantaneous turbidity data acquired over 55 s are averaged and convert it to dry cell concentration, X, every minute. Also, volume of the culture broth, V, is estimated from the averaged output data of weight loss of feed solution reservoir, W, using an electronic balance on which the reservoir is placed. Then, the computer software is used to perform linear regression analyses over the past 30 min of the total biomass, VX, the natural logarithm of the total biomass, ln(VX), and the weight loss, W, in order to calculate volumetric growth rate, d (VX) d t , specific growth rate, μ [= dln (VX) d t ] and the rate of W, d W d t , every minute in a fed-batch culture. The software used to perform the first-order regression analyses of VX, ln(VX) and W was applied to batch or fed-batch cultures of Escherichia coli on minimum synthetic or natural complex media. Sample determination coefficients of the three different variables (VX, ln(VX) and W) were close to unity, indicating that the calculations are accurate. Furthermore, growth yield, Y x s , and specific substrate consumption rate, qsc, were approximately estimated from the data, d (VX) d t and d W d t in a ‘balanced’ fed-batch culture of E. coli on the minimum synthetic medium where the computer-aided substrate-feeding system automatically matches well with the cell growth.

Production of 6-hydroxynicotinic acid, an important starting material for the synthesis of modern pesticides through bacterial position-specific hydroxylation of nicotinic acid, was investigated. Resting cells of Serratia marcescens IFO 12648 were found to catalyze the potential hydroxylation activity of nicotinic acid to produce 6-hydroxynicotinic acid. The optimum culture conditions of S. marcescens IFO 12648 for the accumulation of 6-hydroxynicotinic acid were investigated. The addition to the culture medium of molybdenum and iron ions and of nicotinic acid as an inducer greatly enhanced the hydroxylation activity. Under the optimum conditions, 98.5% of the added 2.2 M nicotinic acid was converted to 6-hydroxynicotinic acid, and the highest yield achieved was 301 g of 6-hydroxynicotinic acid per liter of reaction mixture containing 3.98 g dry weight of resting cells during a 72-h reaction at 35°C.

Membrane-bound nicotinic acid dehydrogenase, an enzyme that catalyzes the formation of 6-hydroxynicotinic acid from nicotinic acid, was solubilized with Triton X-100, and then purified 126-fold with an 11.1% overall recovery from nicotinic acid-induced cells of Pseudomonas fluorescens TN5. The purified enzyme appeared to be homogeneous from analysis by polyacrylamide gel electrophoresis. The enzyme had a molecular mass of approximately 80 kDal and consisted of one subunit. Some electron acceptors, such as phenazine methosulfate, K 3 Fe(CN) 6 and nitro blue tetrazolium, acted as electron acceptors. The purified enzyme catalyzed the hydroxylation of nicotinic acid to 6-hydroxynicotinic acid at a rate of 672 μmol min −1 mg −1 of protein at 35°C. It also catalyzed the hydroxylation of pyrazinecarboxylic acid, 3-pyridinesulfonic acid, and 3-cyanopyridine. The purified enzyme exhibited an optimum pH of 8.3, and was sensitive to thiol reagents such as HgCl 2 and p -chloromercuribenzoate. A reduction in the amount of cytochrome c -like component in the respiratory particles was observed during the hydroxylation reaction of nicotinic acid. Thus, nicotinic acid dehydrogenase appeared to be linked to the cytochrome respiratory chain in the cells of P. fluorescens TN5.

A comparison of three automated feeding strategies was made utilizing on-line turbidity values in fed-batch cultures. Control indices in these feeding strategies were (i) total cell mass (VX), (ii) total cell mass (VX) and estimated cell yield on the basis of the total amount of the substrate supplied ( Y x/lms ), and (iii) growth rate (d(VX)/dt). A marine microorganism, Alteromonas putrefaciens, was cultivated by feeding a solution composed of peptone (220 g/l) and yeast extract (110 g/l). When the automatic feeding strategy (i) was applied, a considerable amount of peptide accumulated in the later phase, indicating ‘overfeeding’ of the natural complex nutrients. The improved feeding strategies (ii) and (iii) enabled the overfeeding to be avoided, resulting in more effective utilization of the nutrients fed. Overall, the maximum cell concentrations were increased more than 1.5 times, while the total amounts of nutrients fed were reduced by a half.

Ethanol-producing yeast cells were entrapped with cross-linked chitosan beads (CCB). In repeated batch culture, the number of yeast cells immobilized with CCB finally reached 1.2 × 1010 cells/ml-beads. While this result was almost the same as with calcium alginate, the ethanol concentration produced with CCB was about 1.5 times higher than that with calcium alginate. The most suitable CCB pore size to immobilize yeast cells was about 10 μm, while to produce ethanol it was approximately 15 μm. CCB modified chemically with the trimethylammonium group had a high capacity to immobilize yeast cells, but the ethanol concentration produced was almost the same as with unmodified CCB. CCB as a carrier had higher yeast cell immobilizing and ethanol production capacities than the commercial carriers SIRAN and FIBERM. In continuous culture, ethanol was produced for thirty days at least at an average concentration of 51 g/l when the dilution rate was 0.1 h−1.

The conversion of phosphatidylcholine (PC) to phosphatidylserine (PS) was examined in the presence of l -serine (3.4 M, solubility of serine) using two phospholipase D (PLD) preparations of Streptomyces origin. A biphasic reaction was carried out at 30°C using an aqueous phase containing both PLD and serine and a solvent (ethyl acetate or diethyl ether) phase containing PC. With an increase in the PC concentration from 17.8 mM to 106.8 mM, the conversion decreased from 85% to 36% in 30 min. The effect of choline inhibition was investigated in two ways—by the addition of choline chloride at low PC concentrations and by removing choline enzymatically or physically during the progress of the reaction at high PC concentrations. The addition of various concentrations of choline chloride decreased the reaction rate, conversion and selectivity. The unreacted PC decreased from 36% to 17.6% and further to 1.6% by the addition of choline oxidase and a mixture of choline oxidase and catalase, respectively. There were no decreases in either the selectivity or the final PS content for the ten repeated batches examined when choline released was washed out from the PLD gels immobilized in Octyl-Sepharose CL-4B after each run.

First, by considering all possible combination of methanol (as a carbon-energy source), peptone (as an organic carbon-nitrogen source), and ammonium sulfate (as an inorganic nitrogen source), five batch cultures of a methanol-assimilating bacterium, Protomonas extorquens , were done to elucidate the cause(s) of pH variations during the microbial cultivations. The batch cultures have been classified into five types in terms of stoichiometric equations of cell growth which involve the elements, C, H, O, and N. The equations explained the pH variation (drop and rise) of the batch cultures on the basis of the consumption and liberation of ammonium ion. Then, six fed-batch cultures using a setpoint of high limit were done by feeding either methanol only or methanol plus peptone. Growth rates could be controlled by the amount of substrate(s) fed per pulse. Supplying peptone in addition to methanol enhanced cell growth. Characteristic differences between pH-stat modal fed-batch cultures using a low limit and those using a high limit, and advantages of the pH-stat modal fed-batch culture using a setpoint of high limit are discussed.

A replica technique was developed to improve the conventional method for screening poly(3-hydroxyalcanoate) [P(3HA)]-accumulating bacterial colonies on agar plates by means of Nile blue A staining. In the conventional method the ethanolic dye solution causes damage to the colonies. This is avoided in the replica technique by transferring a portion of a colony's cells onto a membrane, staining the membrane with a 1% Nile blue A ethanolic solution, and irradiating the dried membrane in the dark UV light. Cells that accumulate P(3HA) exhibit blight-orange fluorescence, whereas cells that do not accumulate P(3HA) appear as dark spots. Glass fiber filter paper was found to be the most suitable membrane material. The effectiveness of the technique was demonstrated using colonies of P(3HA)-accumulating Alcaligenes eutrophus and Alcaligenes latus.