Ryo Ohashi

Continuous production of lactic acid from molasses by perfusion culture of Lactococcus lactis using a stirred ceramic membrane reactor

A perfusion culture system was used for continuous production of lactic acid by retaining cells at a high density of Lactococcus lactis in a stirred ceramic membrane reactor (SCMR). After the cell concentration increased to 248 g/l, half of the culture broth volume was replaced with the fermentation medium. Subsequently, a substrate solution containing glucose (run 1) or molasses (run 2) was continuously supplied to the cells retained in the SCMR. Simultaneously, the culture supernatant was extracted using a ceramic filter with a pore size of 0.2 mum. The dilution rate was initially set at 0.4 h(-1) and gradually decreased to 0.2 h(-1) due to reduction in the permeability of the filter. The concentration of glucose in the substrate solution was adjusted to 60 g/l for the transition and the first period until 240 h, 90 g/l for the second period from 240 h to 440 h, and 70 g/l for the third period from 440 h to 643 h. The average concentration of lactic acid in the filtrate reached 46 g/l in the first period, 43 g/l in the second period, and 33 g/l for the third period. The productivity obtained for the first period reached 15.8 g.l(-1).h(-1), twice as much as that achieved in repeated batch fermentations. Based on the results obtained in run 1, the substrate solution containing 120 g/l of molasses was continuously supplied for 240 h in run 2. The concentration and productivity of lactic acid reached 40 g/l and 10.6 g.l(-1).h(-1), respectively, by continuously replenishing the culture medium at a dilution rate of 0.26 h(-1). These results demonstrated that the filtration capacity of the SCMR was sufficient for a continuous and rapid replenishment of molasses solution from the dense cell culture and, therefore, the perfusion culture system is considered to provide a low-cost process for continuous production of lactic acid from cheap resources.

Initial anchoring and proliferation of fibroblast L-929 cells on unstable surface of calcium phosphate ceramics.

Calcium phosphate ceramics constructed from beta-tricalcium phosphate (TCP) and hydroxyapatite (HAP) have been successfully used as implant materials. However, there is a possibility that these materials are responsible for an unwanted inflammatory response during wound healing. Since TCP is soluble in the body, the instability of this material may contribute to this inflammatory response. Using composite ceramics of TCP and HAP that possessed Ca/P molar ratios of 1.50, 1.55, 1.60, 1.64, and 1.67, the effect of surface instability on fibroblast L-929 cells was investigated. The time-dependent variation of the initial anchoring ratio, cell density, and cell viability were measured. In general, the cells were severely damaged and ruptured on the highly soluble thin surface layer of the TCP-HAP ceramics. The initial anchoring ratio for TCP-HAP ceramics was as high as that for the polystyrene dish (Lux, control). However, viability at 6 h decreased to less than 50% of the initial cell density on ceramics with a Ca/P molar ratio of 1.64 (20% TCP-80% HAP), while 85% of the cells were viable on Lux. The viability on 100% TCP, whose surface is the most highly soluble among the TCP-HAP ceramics used in this study, was reduced to 20%. Morphological observation showed that the anchored cells were ruptured when grown in culture medium on the 100% TCP. Although the high solubility of the thin surface layer on the TCP-HAP ceramics of the carrier was found to be the dominant factor in the decreasing cell viability, the initial viability was enhanced by the stabilization of the surface of the TCP-HAP ceramics by pre-incubating the scaffolds in a culture medium containing 10% fetal bovine serum for 3 d.

Growth and Adhesion of Osteoblast-Like Cells Derived from Neonatal Rat Calvaria on Calcium Phosphate Ceramics

The effects of biocompatible ceramics on the growth and adhesion of osteoblast-rich rat calvarial cell cultures were investigated. Osteoblast-like cells and mouse fibroblast-like L-929 cells were cultured on composite sinters of hydroxyapatite (HAP) and beta-tricalcium phosphate (TCP) culture carriers, whose Ca/P molar ratios were adjusted to values of 1.50, 1.55, 1.60, 1.64 and 1.67. The growth rates of both cell types were accelerated on the TCP-HAP ceramics as compared to those on polystyrene plastic (LUX) or bioinert zirconia ceramics. The population of osteoblast-like cells reached a density of 2.28 x 10(5) cells/cm2 on 100% HAP (Ca/P ratio 1.67) at 9 d of culture, while the corresponding cell density was 1.66 x 10(5) cells/cm2 on LUX and 1.26 x 10(5) cells/cm2 on zirconia. Adhesion of the osteoblast-like cells on TCP-HAP ceramics was similarly increased as compared with that on LUX or zirconia ceramics. The adhesion of L-929 cells on TCP-HAP ceramics was found to be weaker than that on cultures on LUX or zirconia ceramics. The time-dependent variations in the alkaline phosphatase activity of the osteoblast-like cells showed that the osteoblastic phenotype was potentiated by culturing the cells in calcium-rich media. The surface analyses of the Ca/P ratio and the microstructure by XRD and FTIR suggest that the Ca-rich surface was newly formed by recombination on the surface layer in the culture medium containing fetal bovine serum. These results suggest that the surface of TCP-HAP ceramics, especially that of 100% HAP ceramics, are effective for accelerating growth and differentiation of osteoblast-like cells. This is most probably due to the chemical and physical instability and composition of 100% HAP, which promote the formation of a Ca-rich layer at the cell-material interface and provision of Ca ions to the osteoblast-like cells.

Improvement of filtration performance of stirred ceramic membrane reactor and its application to rapid fermentation of lactic acid by dense cell culture of Lactococcus lactis

A ceramic filter was fitted in a stirred ceramic membrane reactor (SCMR) for both extraction of culture supernatant and feeding of distilled water in reverse flow. The dependence of filtration performance on the cell concentration was decreased by about 20% by regularly cleaning the filter using a membrane cleaning system. The improved permeability effected an increase of both the growth rate and viability of Lactococcus lactis by increasing the dilution rate of the culture supernatant. Using the improved SCMR system, a cell concentration of 178 g/l and viability of 98% were obtained after 198 h of culture, while it took 238 h to obtain a cell concentration of 141 g/l and 94% viability without the use of the membrane cleaning system. The perfusion culture system was applied to the rapid batch fermentation of lactic acid by retaining cells at a high density in the SCMR. When the cell concentration reached 80 g/l, the culture supernatant was extracted and replaced with the fermentation medium. Batch fermentation using the retained cells was repeated six times. The concentration of lactic acid increased to more than 30 g/l within 2 h in each fermentation, while 1.2 h was necessary for replacing the culture supernatant to repeat the batch fermentation. The production rate of lactic acid was increased in proportion to the cell concentration, and a high fermentation activity of the retained cells was maintained via the repeated batch fermentation. These results demonstrate that the improved permeability of the SCMR with use of a membrane cleaning system effected a rapid increase in the concentration and viability of cells, and accordingly, the increased production rate of lactic acid in proportion to the concentration of viable cells.

Further biocompatibility testing of silica-chitosan complex membrane in the production of tissue plasminogen activator by epithelial and fibroblast cells

The effects of the physicochemical characteristics of a silica-chitosan complex membrane (SiCM) on the expression of tissue plasminogen activator (tPA) by contacting cells were investigated with the aim of improving the biocompatibility of the novel implant biomaterial. Expression of tPA is considered to be effective in wound healing by preventing thrombus formation, which causes inflammatory responses and rejection of implant materials. Inducing the epithelial cells surrounding implant materials to secrete tPA, which serves as an early signaling system to proliferate cells underlying connective tissues, would be further effective in accelerating wound healing. An epithelial 293 cell line derived from human embryonic kidney and a fibroblast IMR-90 cell line from human lung possessing the ability to secrete tPA were cultured on SiCMs, whose composition was stepwise controlled by adjusting the mixing ratio between silica and chitosan to give silica contents of 20, 33, 43, and 50wt%. Both strains showed strong adhesion on chitosan (0%-SiCM) and 50%-SiCM. The cell proliferation rates were also accelerated in a manner that was dependent on the increase in the adhesion strength of the cells cultured on the SiCMs. Furthermore, the tPA activity in the culture medium increased in accordance with the cell density, while the cellular specific activity of IMR-90 cells to secrete tPA was synergistically enhanced by strong adhesion and a high cell density on the surface of chitosan and 50%-SiCM. Analysis of the physico-chemical effects of the SiCMs revealed that the cells were dominantly affected by the surface hydrophobicity rather than by the zeta potential, as well as by the mixing ratio between chitosan and silica. The wet contact angles of 50%-SiCM and chitosan, which were 68 degrees and 65 degrees , respectively, were found to be suitable for adhesion and growth of both the epithelial 293 cells and fibroblast IMR-90 cells. A hydrophobic surface at 65 degrees -68 degrees was also effective for the production of tPA by IMR-90 cells, whereas the tPA activity of 293 cells reached its highest level on the SiCM with a wet contact angle of 63 degrees . These results suggest that a suitable adhesion strength is a significant factor in the expression of tPA by cells contacting an implant biomaterial.

Continuous Production and Separation of Ethanol without Effluence of Wastewater Using a Distiller Integrated SCM-Reactor System

Abstract Continuous ethanol production without effluence of wastewater was carried out using a closed circulation system which integrated a cell retention culture system and a distillation system to separate ethanol. The stirred ceramic membrane reactor (SCMR), a jar fermentor fitted with asymmetric porous alumina ceramic membrane rods, was used for retaining high density of cells and extraction of the culture supernatant that was continuously sent to the distiller to evaporate ethanol. After the distillation process, the residual solution of the culture supernatant was returned to the SCMR via a heat exchanger. Two types of strains of Saccharomyces cerevisiae ; a flocculating IR2 strain and a sake-brewing K901 strain, were cultured using the integrated fermentation system for continuous production of ethanol. When the ethanol concentration reached 60 g/ l , the culture supernatant was extracted by filtration from the SCMR and sent to the distiller. During the repeated ethanol fermentation and recycling of the medium, cell concentration of K901 strain was increased to 236 g/ l and the productivity of ethanol reached 13.1 g · l −1 · h −1 , which was higher than the productivity of 12.5 g · l −1 · h −1 achieved by immobilized cells on calcium alginate beads (CAB). In addition, the productivity was further increased to 14.5 g · l −1 · h −1 by immobilized K901 cells on CAB retained in the SCMR. In contrast, the flocculating IR2 strain was found to accumulate into crusts of cells on the surface of the ceramic filter and thereby the productivity of ethanol considerably decreased because of the reduced filtration performance. These results showed that the long-lasting and high filtration performance was essential for the increase in the productivity of ethanol using the integrated system that consisted of the SCMR and the distiller.

A dense cell culture system for microorganisms using a shake flask incorporating a porous ceramic filter

Abstract A novel reactor design incorporating a porous ceramic tubular membrane fitted inside a shake flask was developed for the dense cell culture of microorganisms on an ordinary shaker. The tubular membrane was effective in extracting the culture broth. The filtering performance was found to be enhanced by fitting the filter at the shoulder of the flask. Membrane fouling was prevented for long periods by waves created in the culture broth due to the shaking action, which constantly washed the membrane surface. Using this shake flask system, the concentration of Saccharomyces cerevisiae cell mass reached 235 g/ l at 220 h with the feeding of fresh medium and extraction of the supernatant. The long-lasting and high permeability performance of the ceramic membrane enabled increases in the concentration and productivity of viable cells to be readily achieved by continuously refreshing the culture supernatant.

A dense cell culture system for aerobic microorganisms using a shaken ceramic membrane flask with surface aeration

Abstract Aeration of the head space in a shaken ceramic membrane flask (SCM flask) capped with a cotton plug was found to be essential for increasing cell concentration and viability of aerobic microorganisms. Ventilation through the cotton plug cap was insufficient for satisfying the oxygen demand of aerobically growing cells in the SCM flask. Dissolved oxygen concentration (DO) in the culture supernatant of Escherichia coli in batch culture using the SCM flask dropped to nearly 0 ppm when the cell concentration reached 1 g/ l , while CO 2 concentration in the head space of the SCM flask increased quickly to nearly 20% owing to accumulation of the evolved CO 2 . In contrast, the oxygen transfer coefficient in a SCM flask shaken at 230 rpm was as high as 220 h −1 , compared to that in a stirred type jar fermentor. Consequently, aeration of the head space of the SCM flask was found to be sufficient for supplying oxygen by vigorous waves created in the culture broth due to the reciprocal shaking action. Using the SCM flask, E. coli cell mass reached 84 g/ l in 40 h with aeration of the head space with oxygen-enriched gas. The SCM flask with aeration to the head space enabled an increase in the concentration and productivity of viable cells to be readily achieved by continuously replenishing the culture supernatant with oxygen in conjunction with the removal of the evolved CO 2 .

A mini-scale mass production and separation system for secretory heterologous proteins by perfusion culture of recombinant Pichia pastoris using a shaken ceramic membrane flask

The perfusion culture technique using a shaken ceramic membrane flask (SCM flask) was applied to the production of a secretory heterologous protein. A recombinant methylotrophic yeast strain, Pichia pastoris, was cultured aerobically on a reciprocal shaker using an SCM flask. High-level production of human serum albumin (HSA) was attempted by increasing both the cell concentration and the expression level of the recombinant gene. In the two-stage culture method, the cell concentration was first raised to 17 g/l by feeding glycerol, after which the expression of HSA was induced by feeding methanol. However, the concentration of HSA in the effluent filtrate was as low as 0.15 g/l, while the cell concentration continued to increase. In contrast, HSA was effectively produced by feeding methanol from an early stage of the culture. In this case, the HSA concentration reached 0.24 and 0.46 g/l, respectively, using the growth-associated production method without and with aeration into the head space of the SCM flask. The results showed that supplying sufficient oxygen together with the growth-associated induction method are effective for obtaining high-level expression of the methanol-inducible recombinant gene of P. pastoris. An HSA concentration in the filtrate of 1.5 g/l was finally achieved when the cell concentration was increased to 53 g/l by supplying oxygen-enriched gas to the SCM flask. The yield and productivity of HSA reached 2.6-fold and 10-fold those obtained in an ordinary fed-batch culture using a shake flask, and these levels were readily achieved by continuous replenishment of the culture supernatant. The achievements made in this study should contribute to the development of a handy bioreactor system for mini-scale mass production of target proteins with separation at high purity.

High-level expression of the methanol-inducible β-galactosidase gene by perfusion culture of recombinant Pichia pastoris using a shaken ceramic membrane flask

The perfusion culture technique using a shaken ceramic membrane flask (SCM flask) was applied to achieve high-level expression of recombinant gene. A recombinant methylotrophic yeast strain, Pichia pastoris, was cultured aerobically with head space ventilation on a reciprocal shaker using an SCM flask. High-level production of β-galactosidase was attempted by increasing both the cell concentration and the intracellular content of β-galactosidase. The productivity and yield of β-galactosidase were compared between two-stage culture and growth-associated production methods. In the two-stage culture method, the cell concentration was first raised to 57 g/l at 121 h by feeding glycerol and, thereafter, expression of β-galactosidase was induced by feeding methanol. The β-galactosidase activity in the culture broth quickly increased up to 96 kU/ml within 48 h of initiating the production phase, while the cell concentration continued to increase, reaching 106 g/l after 167 h culture. On the other hand, in the growth-associated production method, β-galactosidase was produced from an early stage of the culture by the feeding of methanol. The β-galactosidase activity reached 152 kU/ml at 168 h, while the cell concentration was depressed to 49.7 g/l. The results showed that the growth-associated production method with the feeding of methanol was highly effective for high-level expression of the methanol-inducible recombinant gene of Pichia pastoris. A β-galactosidase productivity level 10 times higher than that obtained in an ordinary fed-batch culture using a shake flask was readily achieved by continuous replenishment of the culture supernatant.