宮永 一彦

We designed a bacteriophage T2 system to display proteins fused at the N-terminus of the head protein small outer capsid (SOC) of a T2 phage. To facilitate selection of chimeric phage, a T2 phage encoding the beta-galactosidase gene (beta gal) upstream of the soc gene was constructed. The phage, named T2 Gal, produces blue plaques on agar plates containing XGal. Subsequently, a plasmid encoding the target protein upstream of soc was constructed and used to transform E. coli B-E cells. Transformed cells were infected with T2 beta Gal and homologous recombination between phage DNA and the plasmid resulted in a chimeric phage that produced transparent plaques due to the excision of the beta gal gene. Chitosanase of Bacillus sp. strain K17 (ChoK), consisting of 453 amino acids, was used as a model target protein. Recombinant T2 phage that produced ChoK was named T2ChoK. T2ChoK was produced from T2 beta Gal at a recombination frequency of about 0.1%. On the other hand, the value for T2 beta Gal produced from wild-type T2 was 0.001%. This new system enables us to select recombinant phage very quickly and accurately. The number of molecules of ChoK was calculated at 14.7 per single phage. Latent period and burst size were estimated for the chimeric phages.

A rapid and simple selection method of high-yield cells has been desired to establish highly productive cell lines for useful secondary metabolites. For this purpose, a new attempt was made to partition cultured plant cells in a poly(ethylene glycol)-dextran aqueous two-phase system (ATPS). The applicability of the ATPS in partitioning cultured strawberry cells (designated FAW) was investigated. The result of single-step partitioning in the ATPS supplemented with 0.4 mmol/kg lithium sulfate showed that FAW cells cultivated for 7 d under light-irradiation were separated into two cell populations with significantly different anthocyanin content. Additionally, the analysis technique of microscopic cell images showed that cells accumulating a high level of anthocyanin were partitioned completely into the bottom phase in a partitioning experiment of FAW cells cultivated for 10 d under light-irradiation in the ATPS supplemented with 1.8 mmol/kg potassium phosphate buffer. These results indicated that cell partitioning in ATPS increased the intracellular anthocyanin content and that the cultured strawberry cell population was heterogeneous in terms of cell surface properties. This is the first report of partitioning based on the heterogeneity of the cell surface properties correlated with the intracellular secondary metabolism in cultured plant cells. Our results also suggested that the ATPS was appropriate as a large-scale method for selecting useful cell lines among the cultured plant cells.

A rapid and simple selection method of high-yield cells has been desired to establish highly productive cell lines for useful secondary metabolites. For this purpose, a new attempt was made to partition cultured plant cells in a poly(ethylene glycol)-dextran aqueous two-phase system (ATPS). The applicability of the ATPS in partitioning cultured strawberry cells (designated FAW) was investigated. The result of single-step partitioning in the ATPS supplemented with 0.4 mmol/kg lithium sulfate showed that FAW cells cultivated for 7 d under light-irradiation were separated into two cell populations with significantly different anthocyanin content. Additionally, the analysis technique of microscopic cell images showed that cells accumulating a high level of anthocyanin were partitioned completely into the bottom phase in a partitioning experiment of FAW cells cultivated for 10 d under light-irradiation in the ATPS supplemented with 1.8 mmol/kg potassium phosphate buffer. These results indicated that cell partitioning in ATPS increased the intracellular anthocyanin content and that the cultured strawberry cell population was heterogeneous in terms of cell surface properties. This is the first report of partitioning based on the heterogeneity of the cell surface properties correlated with the intracellular secondary metabolism in cultured plant cells. Our results also suggested that the ATPS was appropriate as a large-scale method for selecting useful cell lines among the cultured plant cells.

To investigate the therapeutical use of phage mixture for controlling gastrointestinal Escherichia coli O157:H7 cells, in vitro and in vivo experiments were conducted. Three phages, SP15, SP21, and SP22 were selected from 26 phage stock screened from feces of stock animals and sewage influent. Addition of single or binary phage to the E. coli cell batch-culture reduced the turbidity of the culture. However, reascend of the turbidity due to the appearance of phage resistance cell was observed. On the other hand, addition of three phage mixture (SP15-21-22) did not produce reascend of culture turbidity under aerobic condition. Under anaerobic condition, slight reascend of culture turbidity was observed after SP15-21-22 addition. Chemostat continuous culture was operated under anaerobic condition to optimize the titer of phage cocktail and frequency of the addition for controlling E. coli cells. Five-log decrease of E. coli cell concentration after addition of phage cocktail of 10(9) Plaque forming unit (PFU)/ml was observed. However, reascend of cell concentration was observed after 1 d incubation. Repeated addition of phage cocktail was effective to reduce the cell concentration. Suspension of phage cocktail in the buffer containing 0.25% CaCO(3) neutralized 9 times much more buffer of pH 2. Based on this in vitro experiment, phage cocktail (SP15-21-22) suspended in the buffer containing 0.25% CaCO(3) was orally administrated to the mice in which E. coli O157: H7 cells was administrated in 2-d advance. E. coli and phage concentration in the feces was monitored for 9 d after phage addition. High titer of phage was detected in the feces when the phage cocktail administrated daily. E. coli O157: H7 concentration in the feces has been reduced according to the time period. However, difference of E. coli concentration in the feces of mice administrates with phage and in the control mice without phage addition became slight after 9-d test period. High titer of the phage settled down in the gastrointestinal tracts and reduced the concentration of E. coli cell. Repeated oral administration of SP15-21-22 was effective for rapid evacuation of E. coli O157:H7 from the feces and gastrointestinal tract of mice.

Artificial control of phage specificity may contribute to practical applications, such as the therapeutic use of phages and the detection of bacteria by their specific phages. To change the specificity of phage infection, gene products (gp) 37 and 38, expressed at the tip of the long tail fiber of T2 phage, were exchanged with those of PP01 phage, an Escherichia coli O157:H7 specific phage. Homologous recombination between the T2 phage genome and a plasmid encoding the region around genes 37-38 of PP01 occurred in transformant E. coli K12 cells. The recombinant T2 phage, named T2ppD1, carried PP01 gp37 and 38 and infected the heterogeneous host cell E coli O157:H7 and related species. On the other hand, T2ppD1 could not infect E. coli K12, the original host of T2, or its derivatives. The host range of T2ppD1 was the same as that of PP01. Infection of T2ppD1 produced turbid plaques on a lawn of E. coli O157:H7 cells. The binding affinity of T2ppD1 to E. coli O157:H7 was weaker than that of PP01. The adsorption rate constant (k(a)) of T2ppD1 (0.17 x 10(-9) (ml CFU(-1) min(-1)) was almost 1/6 that of PP01 (1.10 x 10(-9) (ml CFU(-1) min(-1))). In addition to the tip of the long tail fiber, exchange of gene products expressed in the short tail fiber may be necessary for tight binding of recombinant phage. (C) 2004 Elsevier B.V. All rights reserved.

As a biochemical approach to reduce the amount of excess sludge from wastewater treatment process, sludge solubilization induced by changing the sludge environment by alternating aerobic and anaerobic operations has been proposed. The sludge solubilization leads to the release of carbonaceous and nitrogenous compounds, which is to be reduced time by time due to the energy consumption by the repeated microbial assimilation. Batch experiments were carried out under various operating conditions alternating aerobic and anaerobic period to characterize the behavior of nitrogenous compounds in the sludge solubilization. Nitrogenous compounds among intracellular matters released by the lysis were successively transformed biochemically, finally having been removed completely under an optimal condition. However, under the conditions out of the optimum, the released nitrogen such as ammonia and nitrate is accumulated in the system. Ammonia accumulation was found to be due to the higher protease activity associated with the protein hydrolysis originated from cell lysis. (C) 2004 Elsevier B.V. All rights reserved.

As a biochemical approach to reduce the amount of excess sludge from wastewater treatment process, sludge solubilization induced by changing the sludge environment by alternating aerobic and anaerobic operations has been proposed. The sludge solubilization leads to the release of carbonaceous and nitrogenous compounds, which is to be reduced time by time due to the energy consumption by the repeated microbial assimilation. Batch experiments were carried out under various operating conditions alternating aerobic and anaerobic period to characterize the behavior of nitrogenous compounds in the sludge solubilization. Nitrogenous compounds among intracellular matters released by the lysis were successively transformed biochemically, finally having been removed completely under an optimal condition. However, under the conditions out of the optimum, the released nitrogen such as ammonia and nitrate is accumulated in the system. Ammonia accumulation was found to be due to the higher protease activity associated with the protein hydrolysis originated from cell lysis. (C) 2004 Elsevier B.V. All rights reserved.

Eucommia ulmoides was cultivated in suspended culture and formed aggregates with broad size spectrum. According to the morphological features, density and size, the aggregates were classified into daughter aggregate (DA), meristemoid-like aggregate (MA) and old aggregate (OA). DA can grow into MA, which proliferates DAs again at some vigorously growing points on their surface. Coexistence of DAs and MAs implies better conditions for callus growth. It was revealed that mechanical disintegration of callus into smaller size in the subculture has a negative effect on callus growth due to the damage of active cells located at the aggregate surface and/or the characteristics of original big aggregate which belongs to OA before fragmentation. The growth rates of spontaneous DA and MA were about two times higher than that of the disintegrated callus. As an approach for scaling-up process, spontaneously proliferated aggregates can provide the rapidly growing callus of E. ulmoides culture compared with the mechanically disintegrated callus. (C) 2004 Elsevier B.V. All rights reserved.

Escherichia coli has been used as an indicator of the fecal contamination of water and food, identifying potential health hazards. In this study, an E coli-specific bacteriophage, T4, was used to detect E. coli bacteria. The T4 phage small outer capsid (SOC) protein was used to present green fluorescent protein (GFP), an easily detectable marker protein, on the phage capsid. To inactivate phage lytic activity, we used the T4e(-) phage, which does not produce the lysozyme responsible for host cell lysis. Infection of E. coli K12 cells with the GFP-labeled T4e(-) phage (T4e(-)/GFP) enabled the visualization and distinction of E coli K12 cells from T4 phage-insensitive cells, Pseudomonas aeruginosa. Prolonged incubation of E. coli K12 cells with the T4e(-)/GFP phage did not lead to cell lysis. Propagation of T4e(-)/GFP in host cells increased the intensity of green fluorescence, making the distinction of E coli cells from other cells simple and effective. This method enables the rapid, conclusive quantitation of E. coli cells within an hour. (C) 2004 Elsevier B.V. All rights reserved.

Escherichia coli has been used as an indicator of the fecal contamination of water and food, identifying potential health hazards. In this study, an E coli-specific bacteriophage, T4, was used to detect E. coli bacteria. The T4 phage small outer capsid (SOC) protein was used to present green fluorescent protein (GFP), an easily detectable marker protein, on the phage capsid. To inactivate phage lytic activity, we used the T4e(-) phage, which does not produce the lysozyme responsible for host cell lysis. Infection of E. coli K12 cells with the GFP-labeled T4e(-) phage (T4e(-)/GFP) enabled the visualization and distinction of E coli K12 cells from T4 phage-insensitive cells, Pseudomonas aeruginosa. Prolonged incubation of E. coli K12 cells with the T4e(-)/GFP phage did not lead to cell lysis. Propagation of T4e(-)/GFP in host cells increased the intensity of green fluorescence, making the distinction of E coli cells from other cells simple and effective. This method enables the rapid, conclusive quantitation of E. coli cells within an hour. (C) 2004 Elsevier B.V. All rights reserved.

Twenty six phages infected with Escherichia coli O157:H7 were screened from various sources. Among them, nine caused visible lysis of E. coli O157:H7 cells in LB liquid medium. However, prolonged incubation of E. coli cells and phage allowed the emergence of phage-resistant cells. The susceptibility of the phage-resistant cells to the nine phages was diverse. A rational procedure for selecting an effective cocktail of phage for controlling bacteria was investigated based on the mechanism of phage-resistant cell conversion. Deletion of OmpC from the E. coli cells facilitated the emergence of cells resistant to SP21 phage. After 8 h of incubation, SP21-resistant cells appeared. By contrast, alteration of the lipopolysaccharide (LPS) profile facilitated cell resistance to SP22 phage, which was observed following a 6-h incubation. When a cocktail of phages SP21 and SP22 was used to infect E. coli O157:H7 cells, 30 h was required for the emergence of cells (R-C) resistant to both phages. The R-C cells carried almost the same outer membrane and LPS components as the wild-type cells. However, the reduced binding ability of both phages to R-C cells suggested disturbance of phage adsorption to the R-C surface. Even though R-C cells resistant to both phages appeared, this work shows that rational selection of phages has the potential to at least delay the emergence of phage resistance. © Springer-Verlag 2003.

Twenty six phages infected with Escherichia coli O157:H7 were screened from various sources. Among them, nine caused visible lysis of E. coli O157:H7 cells in LB liquid medium. However, prolonged incubation of E. coli cells and phage allowed the emergence of phage-resistant cells. The susceptibility of the phage-resistant cells to the nine phages was diverse. A rational procedure for selecting an effective cocktail of phage for controlling bacteria was investigated based on the mechanism of phage-resistant cell conversion. Deletion of OmpC from the E. coli cells facilitated the emergence of cells resistant to SP21 phage. After 8 h of incubation, SP21-resistant cells appeared. By contrast, alteration of the lipopolysaccharide (LPS) profile facilitated cell resistance to SP22 phage, which was observed following a 6-h incubation. When a cocktail of phages SP21 and SP22 was used to infect E. coli O157:H7 cells, 30 h was required for the emergence of cells (R-C) resistant to both phages. The R-C cells carried almost the same outer membrane and LPS components as the wild-type cells. However, the reduced binding ability of both phages to R-C cells suggested disturbance of phage adsorption to the R-C surface. Even though R-C cells resistant to both phages appeared, this work shows that rational selection of phages has the potential to at least delay the emergence of phage resistance.

Staining of esterase-active bacteria with carboxyfluorescein diacetate ( CFDA) has been used to evaluate the viability of various types of cell. However, the outer membrane of Gram-negative bacteria prevents CFDA from permeating into the cell. Although EDTA can increase the permeability of the outer membrane allowing CFDA to enter the cells, it was experimentally confirmed that there is still considerable difficulty in visualizing viable cells due to passive diffusion of carboxyfluorescein (CF), a hydrolyzed product of CFDA, out of the cells. We found that glutaraldehyde enhances the discriminative recognition of esterase-active Gram-negative bacteria under microscopic observation by improving the efficacy of staining. We believe the successful staining in the presence of glutaraldehyde is due to two separate effects: an increase in the permeability of CFDA into the cell and prevention of leakage of CF out of the cell.

Staining of esterase-active bacteria with carboxyfluorescein diacetate ( CFDA) has been used to evaluate the viability of various types of cell. However, the outer membrane of Gram-negative bacteria prevents CFDA from permeating into the cell. Although EDTA can increase the permeability of the outer membrane allowing CFDA to enter the cells, it was experimentally confirmed that there is still considerable difficulty in visualizing viable cells due to passive diffusion of carboxyfluorescein (CF), a hydrolyzed product of CFDA, out of the cells. We found that glutaraldehyde enhances the discriminative recognition of esterase-active Gram-negative bacteria under microscopic observation by improving the efficacy of staining. We believe the successful staining in the presence of glutaraldehyde is due to two separate effects: an increase in the permeability of CFDA into the cell and prevention of leakage of CF out of the cell.

In a fluidized-bed porous carrier system, organic carbon and nitrogen in wastewater can be removed simultaneously. However, the excess sludge generated in the system has not been focused on. On the other hand, a membrane system can separate suspended solid from treated water and retain slowly growing microbes such as nitrifiers in the aeration tank. Porous carrier and membrane hybrid system with these respective advantages has been studied to enhance the nitrogen removal efficiency. In this study, the hybrid system was investigated to compare with a conventional carrier system in terms of the nitrogen removal rate from complex artificial wastewater (CAW) containing polypeptone as a nitrogen source. Moreover, the amounts of excess sludge generated in both systems were evaluated. Longer sludge retention time of the hybrid system was expected to enhance the microbial autolysis and lead to the reduction of the excess sludge. Consequently, the amount of excess sludge generated in the hybrid system was reduced to about one-fourth of that in the carrier system for about two months. In addition, a lot of slowly growing protozoa preyed on the microorganisms, which might have contributed to the reduction of excess sludge. The removal rates of organic carbon and nitrogen in the hybrid system were higher than in the carrier system, due to the highly concentrated microorganisms in the hybrid system. A batch operation revealed that the nitrogenous compounds of polypeptone in CAW were rapidly removed by assimilation, while urea in the artificial wastewater was mainly removed by nitrification. Consequently, total nitrogen concentration in CAW containing polypeptone decreased faster than in artificial wastewater containing urea. These results show that nitrogenous compounds in CAW are easy to be removed. The hybrid system is an efficient system to enhance the nitrogen removal and has a capability to reduce the excess sludge.

In a fluidized-bed porous carrier system, organic carbon and nitrogen in wastewater can be removed simultaneously. However, the excess sludge generated in the system has not been focused on. On the other hand, a membrane system can separate suspended solid from treated water and retain slowly growing microbes such as nitrifiers in the aeration tank. Porous carrier and membrane hybrid system with these respective advantages has been studied to enhance the nitrogen removal efficiency. In this study, the hybrid system was investigated to compare with a conventional carrier system in terms of the nitrogen removal rate from complex artificial wastewater (CAW) containing polypeptone as a nitrogen source. Moreover, the amounts of excess sludge generated in both systems were evaluated. Longer sludge retention time of the hybrid system was expected to enhance the microbial autolysis and lead to the reduction of the excess sludge. Consequently, the amount of excess sludge generated in the hybrid system was reduced to about one-fourth of that in the carrier system for about two months. In addition, a lot of slowly growing protozoa preyed on the microorganisms, which might have contributed to the reduction of excess sludge. The removal rates of organic carbon and nitrogen in the hybrid system were higher than in the carrier system, due to the highly concentrated microorganisms in the hybrid system. A batch operation revealed that the nitrogenous compounds of polypeptone in CAW were rapidly removed by assimilation, while urea in the artificial wastewater was mainly removed by nitrification. Consequently, total nitrogen concentration in CAW containing polypeptone decreased faster than in artificial wastewater containing urea. These results show that nitrogenous compounds in CAW are easy to be removed. The hybrid system is an efficient system to enhance the nitrogen removal and has a capability to reduce the excess sludge.

For the development of a sewer pipe that removes dissolved organic carbon (DOC) and nitrogen, a rectangular channel with a fabricated porous ceramic bed was investigated. The experimental channel was 1 m long, 2.0 × 10-2 m wide, and 2.5 × 10-2 m deep, the total volume being 0.811. Blocks of the ceramic bed with 2.0 × 10-2 m2 total surface area and 5.0 × 10-3 m thickness were installed along the channel bed for the immobilization of microorganisms. Synthetic wastewater was used as the model sewage. Removal of organic carbon and nitrogen was carried out with or without aeration. Dissolved oxygen (DO) and pH microelectrodes were employed to analyze structure of the biofilm, which played an important role in the removal of both organic carbon and nitrogen. The results give evidence that the combination of aeration and biofilm development enhanced the simultaneous removal of organic carbon and nitrogen. Microelectrode study revealed that the biofilm was rough and heterogeneous in both vertical and horizontal directions and had an average thickness of 2.5-5 mm. The biofilm, consisting of an aerobic/anaerobic layer, was suggested to be responsible for the nitrification/denitrification process, while the aeration accelerated the removal of total organic carbon (TOC), NH4 or NO2 from the model sewage. Sequential nitrification/denitrification proceeded in the biofilm even when aeration was carried out. This study suggests that the immobilization of microbes inside the sewer pipe may be effective for the simultaneous removal of organic carbon and nitrogen in the sewage line. © 2002 Elsevier Science B.V. All rights reserved.

For the development of a sewer pipe that removes dissolved organic carbon (DOC) and nitrogen, a rectangular channel with a fabricated porous ceramic bed was investigated. The experimental channel was 1 m long, 2.0 x 10(-2) m wide, and 2.5 x 10(-2) m deep, the total volume being 0.811. Blocks of the ceramic bed with 2.0 x 10(-2) m(2) total surface area and 5.0 x 10(-3) m thickness were installed along the channel bed for the immobilization of microorganisms. Synthetic wastewater was used as the model sewage. Removal of organic carbon and nitrogen was carried out with or without aeration. Dissolved oxygen (DO) and pH microelectrodes were employed to analyze structure of the biofilm, which played an important role in the removal of both organic carbon and nitrogen. The results give evidence that the combination of aeration and biofilm development enhanced the simultaneous removal of organic carbon and nitrogen. Microelectrode study revealed that the biofilm was rough and heterogeneous in both vertical and horizontal directions and had an average thickness of 2.5-5 mm. The biofilm, consisting of an aerobic/anaerobic layer, was suggested to be responsible for the nitrification/denitrification process, while the aeration accelerated the removal of total organic carbon (TOC), NH4 or NO2 from the model sewage. Sequential nitrification/denitrification proceeded in the biofilm even when aeration was carried out. This study suggests that the immobilization of microbes inside the sewer pipe may be effective for the simultaneous removal of organic carbon and nitrogen in the sewage line. (C) 2002 Elsevier Science B.V All rights reserved.

Porous carriers in fluidized bed have been used for microbial immobilization in order to simultaneously remove organic carbon and nitrogen in wastewater. In particular, multifunctional microbial reactions in the carrier, such as simultaneous nitrification/denitrification, play important roles in nitrogen removal. To enhance these reactions the substrates should be supplied into the carrier with appropriate rates. This is because the denitrification reaction was often limited by the supply of organic substances, due to overgrowth of heterotrophs in the region near the carrier surface. A porous carrier-membrane hybrid process was found to have improved nitrogen removal efficiency, due to stimulated denitrification as well as nitrification. The hybrid system achieved a 30% higher nitrogen removal ratio than that in the fluidized porous carrier system. Microelectrode studies revealed that although intracarrier denitrification rate in a conventional fluidized bed was limited by organic carbon, this limitation was reduced in the hybrid process, resulting in the increased intracarrier denitrification rate. These effects were due to suppressed glucose oxidation in aerobic region in the hybrid process. (C) 2002 Elsevier Science B.V. All rights reserved.

Porous carriers in fluidized bed have been used for microbial immobilization in order to simultaneously remove organic carbon and nitrogen in wastewater. In particular, multifunctional microbial reactions in the carrier, such as simultaneous nitrification/denitrification, play important roles in nitrogen removal. To enhance these reactions the substrates should be supplied into the carrier with appropriate rates. This is because the denitrification reaction was often limited by the supply of organic substances, due to overgrowth of heterotrophs in the region near the carrier surface. A porous carrier-membrane hybrid process was found to have improved nitrogen removal efficiency, due to stimulated denitrification as well as nitrification. The hybrid system achieved a 30% higher nitrogen removal ratio than that in the fluidized porous carrier system. Microelectrode studies revealed that although intracarrier denitrification rate in a conventional fluidized bed was limited by organic carbon, this limitation was reduced in the hybrid process, resulting in the increased intracarrier denitrification rate. These effects were due to suppressed glucose oxidation in aerobic region in the hybrid process. (C) 2002 Elsevier Science B.V. All rights reserved.

Callus induced from the leaves of Eucommia ulmoides, a medicinal woody plant, was cultivated to produce a factor capable of enhancing collagen synthesis in animal cells (FECS). However, the callus was too rigid to be divided into small pieces by a hydrodynamic force during the cultivation, which led to a slow callus growth characterized by enlargement of the callus size rather than increase in the callus number. Improved growth rate of the callus with smaller sizes and cavity formation in the central region of the callus with its enlargement, implied the occurrence of transfer limitation of nutrient(s) inside the callus. Distributions of the principal nutrients of sugar, nitrogen and dissolved oxygen concentrations across the cultivated callus were simulated by a kinetic model consisting of nutrients diffusion and bioreaction kinetics, suggesting that oxygen transfer in the callus was the Limiting factor for the callus growth. A callus growth model considering the effect of the nutrients' transfer and cell death kinetics in the callus which was caused by the oxygen depletion successfully described the callus enlargement process. Based on these results, a newly developed bioreactor with a fragmentation device enabled the callus to grow with enhanced growth rate by controlling the callus at small sizes during the cultivation. (C) 2001 IMACS. Published by Elsevier Science B.V. All rights reserved.

A suspension culture of tobacco cells was transformed with a gene encoding barley lectin to obtain an efficient production system of lectin. Lectin excreting transgenic tobacco was constructed by transformation with barley lectin deleted of the C- terminal vacuole targeting peptide (CTPP). 2,4Dichlorophenoxyacetic acid (2,4- D) was examined for its effect on the excretion of lectin at different concentrations from 0 to 5 mg 1 2,4- D addition increased excretion efficiency, defined as the amount of excreted lectin per cell, but reduced the growth rate. Microscopic observations showed loosening of the cell wall, which is assumed to be one of the causes of the enhanced excretion. Decrease in extracellular lectin was observed in spite of the growth of cells, which was confirmed to be caused by a degradation process in the culture medium. These results suggest that an efficient production process of lectin would include a process of simultaneously removing culture medium with high lectin content before degradation occurs and maintaining the cells at high excretion efficiency.

Cultured plant cells are often highly heterogeneous in terms of secondary metabolite production. We have developed a quantitative determination method that uses an image processing system to estimate such individual cell characteristics as content of the secondary metabolite, anthocyanin. In this study, strawberry cells producing anthocyanins were grown in modified Linsmaier-Skoog medium. Anthocyanin accumulation profiles of individual cells depended on medium compositions and were quantitatively determined using the new method. The modified medium supplemented with riboflavin and high sugar concentration showed a markedly higher anthocyanin accumulation profile and pigmented cell ratio than the other modified media. The maximum content was about 11 mg (g-fresh cell weight)(-1) which was three times higher than that in the control medium. Moreover, the anthocyanin accumulation profiles in the individual cells cultured in all modified media could be approximated to the parts of the normal distribution curves with the constant variance. (C) 2000 Elsevier Science S.A. All rights reserved.

Anthocyanin accumulation in strawberry (Fragaria ananassa) cells cultured on a solid medium was monitored using an image-processing system that did not require direct sampling or destruction of the cells. Because of the intercellular heterogeneity of secondary metabolite production in plant cell cultures, the maximum metabolite concentration in individual cells is often more than 10 times higher than that of the average concentration. An image-processing based method enabled the growth and the pigmentation behavior of individual cells to be traced. Changes in the time courses of the anthocyanin content of individual cells differed from each other, although the average anthocyanin contents increased gradually with time in a batch culture. However, these various changing patterns in the anthocyanin content of each cell were independent of the cell cycle. In addition, image analysis revealed that the two cells just after cell division were almost identical to each other both in size and anthocyanin content. The proposed method which uses an image-processing system provides a useful tool for analyzing the secondary metabolism in individual cultured plant cells.

Anthocyanin accumulation in strawberry (Fragaria ananassa) cells cultured on a solid medium was monitored using an image-processing system that did not require direct sampling or destruction of the cells. Because of the intercellular heterogeneity of secondary metabolite production in plant cell cultures, the maximum metabolite concentration in individual cells is often more than 10 times higher than that of the average concentration. An image-processing based method enabled the growth and the pigmentation behavior of individual cells to be traced. Changes in the time courses of the anthocyanin content of individual cells differed from each other, although the average anthocyanin contents increased gradually with time in a batch culture. However, these various changing patterns in the anthocyanin content of each cell were independent of the cell cycle. In addition, image analysis revealed that the two cells just after cell division were almost identical to each other both in size and anthocyanin content. The proposed method which uses an image-processing system provides a useful tool for analyzing the secondary metabolism in individual cultured plant cells.

Plant-cultured cells are often highly heterogeneous in secondary metabolite productivity. The industrial application for large-scale metabolite production requires establishment of a stable high-producing cell line. In this study, image analysis of the individual cell is investigated as a method for evaluation of a heterogeneous cell population, and compared with the conventional method of estimation, which is based on average-cell productivity. Among strawberry cells producing anthocyanins, cells with a wide-range of pigment concentration were observed and maximum anthocyanin content was 10 times higher than the average value. In addition, a change of the frequency distribution was revealed in batch cultivation. (C) 2000 John Wiley & Sons, Inc.

Plant-cultured cells are often highly heterogeneous in secondary metabolite productivity. The industrial application for large-scale metabolite production requires establishment of a stable high-producing cell line. In this study, image analysis of the individual cell is investigated as a method for evaluation of a heterogeneous cell population, and compared with the conventional method of estimation, which is based on average-cell productivity. Among strawberry cells producing anthocyanins, cells with a wide-range of pigment concentration were observed and maximum anthocyanin content was 10 times higher than the average value. In addition, a change of the frequency distribution was revealed in batch cultivation. (C) 2000 John Wiley & Sons, Inc.

現在の塩化ビニルモノマー(VCM)製造工程においては様々な含塩素有機化合物が副生し、燃焼あるいは活性汚泥などによって処理されている。プロセス内ゼロエミッションのためにば副生成物の分離や物質変換に関わる新たな要素技術の開発が求められる。微生物反応を利用する塩素化エチレンの嫌気的脱塩素化は、生成物をポリ塩化ビニル(PVC)製造の原料として再利用できることが期待できる。製品寿命を終えたPVCの多くは燃焼か埋め立て処分される。PVCの選択的脱塩素化で塩化水素に変換できれば、塩化第二鉄やポリ塩化アルミニウムの原料として利用できる。このような異種プロセス間への新たな物質フローを構築すれば塩化ナトリウムの電気分解に始まり、製品寿命を終えると再び塩化ナトリウムに戻る一連の塩素フローの中で、製品要素としての塩素の寿命を長く設定することができ、新たな電気分解による塩素の製造量を減らすことにつながる。 一方、塩化ナトリウムの電気分解で生成する塩素を利用するためには、バランス産業と呼ばれるように同時に生成するカセイソーダ、水素ガスの生成量に見合う利用プロセスを構築するのが理想的である。しかし日本においてはPVC製造のために必要な塩素のバランスをとることはできず、含塩素半製品輸入によって調整している。種々の添加物を必要とする軟質塩化ビニルを非塩素系の材料に転換することは技術的に可能である。この転換によって、現在の塩素需要過剰状態は解消され、バランス型にすることができる。これを可能にするためには、経済的に可能な種々の技術開発が求められる。

Fragaria ananassa (strawberry) callus, which produced high amounts of anthocyanin in the dark, was isolated from a cell line not producing anthocyanin. The isolated callus (FAR) was homogeneous and more than 90% of the cells were pigmented. The FAR callus accumulated more than 1000 mu g of anthocyanin per g fresh cell in the dark. Four different basal solid media were examined to maintain FAR callus: Though growth rate and anthocyanin concentration were different on each media, total anthocyanin production was about the same at 400 mu g anthocyanin/0.1 g fresh cell wt after 22 days. This FAR cell line could therefore be used for the industrial production of anthocyanin.

Fragaria ananassa (strawberry) callus, which produced high amounts of anthocyanin in the dark, was isolated from a cell line not producing anthocyanin. The isolated callus (FAR) was homogeneous and more than 90% of the cells were pigmented. The FAR callus accumulated more than 1000 mu g of anthocyanin per g fresh cell in the dark. Four different basal solid media were examined to maintain FAR callus: Though growth rate and anthocyanin concentration were different on each media, total anthocyanin production was about the same at 400 mu g anthocyanin/0.1 g fresh cell wt after 22 days. This FAR cell line could therefore be used for the industrial production of anthocyanin.