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Continuous cultures of immobilized Streptomyces kasugaensis, a kasugamycin producer, were carried out on Celite beads. When using a prototype separator for immobilized-cell separation and recycling, the continuous operation could not be sustained for an extended period as a result of an excessive loss of immobilized cells caused by the poor performance of the separator. Accordingly, the immobilized-cell separator was revised to provide better immobilized-cell settling and thus recycling into the reactor. In a subsequent culture using the revised separator, a stable operation was maintained for over 820 h with a high kasugamycin productivity. The kasugamycin productivity ranged from 9.8 to 16.1 mg/L/h, which was about 14- to 23-fold higher than that in a batch suspended-cell culture. When the original feeding medium concentration was doubled at the end of the continuous culture, the productivity became severely impaired for several reasons, which will be discussed. An excessive formation of free cells and loss of immobilized cells through the separator were also observed.

For the production of oil-desulfurizing biocatalyst, a two-stage fermentation strategy was adopted, in which the cell growth stage and desulfurization activity induction stage were separated. Sucrose was found to be the optimal carbon source for the growth of Gordonia nitida CYKS1. Magnesium sulfate was selected to be the sulfur source in the cell growth stage. The optimal ranges of sucrose and magnesium sulfate were 10−50 and 1−2.5 g L^-1, respectively. Such a broad optimal concentration of sucrose made the fed-batch culture easy, while the sucrose concentration was maintained between 10−20 g L^-1 in the actual operation. As a result, 92.6 g L^-1 of cell mass was acquired by 120 h of fed-batch culture. This cell mass was over three times higher than a previously reported result, though the strain used was different. The desulfurization activity of the harvested cells from the first stage culture was induced by batch cultivation with dibenzothiophene as the sole sulfur source. The optimal induction time was found to be about 4 h. The resting-cell biocatalyst made from the induced cells was applied for the deep desulfurization of a diesel oil. It was observed that the sulfur content of the diesel oil decreased from 250 mg-sulfur L-oil^-1 to as low as 61 mg-sulfur L-oil^-1 in 20 h. It implied that the biocatalyst developed in this study had a good potential to be applied to a deep desulfurization process to produce ultra-low-sulfur fuel oils.

Biosynthesis of kasugamycin could be greatly enhanced by applying a nonnutritional stress of pH shock, that is, sequential pH changes from a neutral pH to an acidic condition and then back to the neutral condition. During the acidic period, cell growth decreased to nil. After recovery of the neutral condition, the cell growth resumed after a time lag concurrently with the biosynthesis of kasugamycin at a greatly enhanced rate compared with the control case without a pH shock. In a series of experiments performed to identify the optimal length of pH shock, four different lengths (6, 12, 24, and 48 h) of pH shock were applied. The best result was obtained when pH shock was applied for 24 h, with kasugamycin productivity approximately 7-fold higher than that of the control.

For economic recovery of poly(3-hydroxybutyrate) (PHB) from culture broths of Ralstonia eutropha containing PHB, Al-based and Fe-based coagulants were used in the pretreatment step. The coagulated cells were then separated by centrifugation, and PHB was extracted by chemical digestion with a sodium hypochlorite/chloroform dispersion solution. The practical upper limits of dosage were found to be 1,500 mg-Al/L and 1,000 mg-Fe/L, respectively, for Al- and Fe-based coagulants. When the harvested cells were treated with a 50% sodium hypochlorite/chloroform dispersion solution, PHB recovery and purity were 90−94% and 98−99%, respectively. The influence of the use of coagulants on the PHB recovery process was found to be insignificant. Despite the residual Al and Fe in the recovered PHB (less than 450 mg-Al/kg-PHB and 750 mg-Fe/kg-PHB, respectively), no detectable amounts of Al and Fe were leached from films made of the recovered PHB under acidic conditions. The use of Fe-based coagulants is less recommended because the Fe impurity can cause an unwanted colorization problem in the final product.

Alcaligenes eutrophus containing intracellular poly(3-hydroxybutyrate) was recovered from fermentation broth by centrifugation and filtration after pretreatment with Al- and Fe-based coagulants. Coagulation efficiency was largely affected by pH, and the optimum pH's for cell recovery were about 4.6−5.6 for the Al-based coagulants and about 5−8 for the Fe-based coagulants. Ammonium ions that combined with metals to form complex compounds increased the coagulant requirement, and the additional requirement of coagulant was found to be proportional to the ammonium concentration. In addition, various ligands in addition to ammonium ions contained in the culture medium interfered with the coagulation reaction and increased the coagulant requirement also. The coagulant requirement increased with the cell concentration regardless of coagulant type. The polymeric coagulants such as PACS, Hi-PAX, and Ferix-3 were more effective than nonpolymeric coagulants of aluminum sulfate and ferrous sulfate. The optimum dosages of the coagulants tested were determined over a broad range of cell concentration of 20.5−210 g/L. It was observed that the energy requirement for centrifugation could be greatly reduced with cell coagulation.

An efficient cell-loaded biosupport separator of the decantor type was developed and applied for a continuous perfusion culture to produce cyclosporin A (CyA), in which fungal cells were immobilized on Celite beads. In the preliminary experiments employing highly viscous polymer (carboxymethyl cellulose) solutions, the decantor showed good separation performances at high solution viscosites and dilution rates. Two concentric cylindrical tubes installed inside the decantor turned out to play key roles in the efficient separation of the immobilized cells. By installing the decantor on an immobilized-cell perfusion bioprocess system, a stable continuous operation was possible even at a high dilution rate for an extended period, leading to high productivities of free cells and CyA. Almost no immobilized biomass existed in the effluent stream from the bioreactor, demonstrating the effectiveness of the decantor system. It is noteworthy that we could obtain these results despite unfavorable fermentation conditions, i.e., reduced apparent density of cell-loaded beads and increased drag force on the bead particles caused by overgrowth of cells on the bead surface, tubulence caused by large air bubbles, and the existence of a high density of suspended fungal cells (10 g/L) in the fermentation broth.

In batch cultures for l-ornithine production in which dissolved oxygen concentration and pH were closely controlled, time changes of redox potential were observed in connection with the profiles of cell, glucose, and ornithine concentrations. It was found that the redox potential profile had four different phases reflecting the physiological state of the culture and that it was closely related to cell concentration change. Effects of glucose and ornithine on the redox potential were identified in a separate series of experiments. On the basis of the experimental results, a correlation of redox potential to glucose, cell, and ornithine concentrations has been proposed. The proposed correlation can be used for on-line estimation of ornithine concentration from on-line data of redox potential, glucose concentration, and cell concentration.

A dibenzothiophene (DBT)-degrading bacterial strain was isolated from dyeing industry wastewater and identified as Nocardia sp. CYKS2. The newly isolated bacterial strain Nocardia sp. CYKS2 was able to convert DBT to 2-hydroxybiphenyl (2-HBP) as the dead-end metabolite through a sulfur-specific pathway. Other organic sulfur compounds, such as thiophene derivatives, thiazole derivatives, sulfides, and disulfides were also desulfurized by Nocardia sp. CYKS2. In batch culture, 0.2 mM DBT was completely desulfurized in 60 h. After DBT was depleted, neither cell growth nor 2-HBP production was observed. When a model oil which DBT was dissolved in hexadecane was treated with growing cells, DBT was desulfurized from 10 mM to about 2 mM in 80 h. In this case, desulfurization rate was 0.279 mg-sulfur/(L-dispersion·h), which was about 2.5 times higher than that in the previous case of batch culture. When diesel oil was treated, the sulfur content decreased from 0.3 to 0.24 wt % in 48 h. A volumetric phase ratio of oil to water was 1/10 in this case. The sulfur decreased from 0.3 to 0.2 wt % in 48 h, when the volumetric phase ratio was 1/20. The desulfurization rates were 0.909 and 0.992 mg-sulfur/(L-dispersion·h), respectively.

An efficient sporulation/immobilization procedure for immobilized fungal cell culture was developed by modifying an existing immobilized technique to shorten the time and number of steps for sporulation. This method was applied to an immobilized-cell perfusion bioprocess (IPB) for continuous production of CyA, an intracellular secondary metabolite produced by a filamentous fungus, Tolypocladium inflatum. In the IPB, the fungal cells were immobilized in the pores of celite beads (100~500 μm) and a top-driven stirred tank fermentor was used for the culture. The IPB showed good process benefits as demonstrated by the high density of immobilized cells continuously producing CyA-containing free cells. The productivity of CyA-containing free cells in the effluent was very high, ca. 1.0 g/(L/h) at a dilution rate of 0.1 h^-1, due to the high density of immobilized cells in the fermentor. The CyA productivity was 4.0~6.0 mg/(L/h) which was about 6~10-fold higher than that of batch suspended cell culture. Such an efficient IPB was possible since a decantor was developed in this study, which could effectively separate cell-immobilized beads from the effluent although bead loss slightly increased as the cell loading increased in the latter part of culture. Furthermore, long-term operation of IPB was carried out successfully by employing an in-situ immobilization strategy. It was found that a large number of spores in the fermentation broth in the reactor were entrapped in-situ into the newly supplemented celite beads and then germinated, thus forming new immobilized cells.

Continuous culture for the production of ethanol from wood hydrolysate was carried out in an internal membrane-filtration bioreactor. The hydrolysate medium was sterilized at a relatively low temperature of 60 °C with the intention of reducing the formation of inhibitory compounds during the sterilization. The maximum ethanol concentration and productivity obtained in this study were 76.9 g/L and 16.9 g/L-h, respectively, which were much higher than those (57.2−67 g/L and 0.3−1.0 g/L-h) obtained in batch cultures using hydrolysate media sterilized at 60 °C. The productivity was also found to be much higher than that (6.7 g/L-h) obtained in a continuous cell retention culture using a wood hydrolysate sterilized at 121 °C. These results show that the internal membrane-filtration bioreactor in combination with low-temperature sterilization could be very effective for ethanol production from wood hydrolysate.