Ferda Mavituna

Effect of perfluorodecalin as an oxygen carrier on actinorhodin production by Streptomyces coelicolor A3(2)

Perfluorodecalin, a perfluorocarbon (PFC), was used in this investigation as a dissolved oxygen carrier in the media of Streptomyces coelicolor cultures. The effects of different concentrations of PFC, PFC emulsified with pluronic F-68 and pluronic alone were investigated in the shake-flask cultures using both defined and complex media. In the defined medium with PFC alone, the maximum biomass and actinorhodin concentrations and the volumetric substrate consumption rates increased with increasing PFC concentration. They decreased dramatically, however, when the PFC concentration exceeded 50% (v/v). Emulsifying the PFC with pluronic F-68 resulted in a significant increase in antibiotic concentration while growth was unaffected. The inclusion of more than 4 g/l pluronic alone in the fermentation medium inhibited the growth. In the complex medium with 40% (v/v) PFC, although the final antibiotic concentration was unaffected, the onset of actinorhodin accumulation was 2 days earlier than that in the control. It was demonstrated that PFC and emulsified PFC did not have any deleterious effects on S. coelicolor cultures.

Growth of immobilised plant cells in reticulate polyurethane foam matrices

SummaryAn inoculum of initially freely suspended cell aggregates ofCapsicum frutescens was immobilised in porous polyurethane foam matrices. Subsequent growth and substrate consumption of these immobilised cells in batch culture were measured and compared with those of suspension cultures. The results showed that the maximum specific growth rate of freely suspended cells was slightly higher than that of immobilised cells but the overall growth patterns and final cell yields were similar.

Somatic embryogenesis and plant regeneration of pepper in liquid media

A protocol was developed for regeneration of pepper (Capsicum annuum var. Ace) through somatic embryogenesis in liquid media. For embryogenic callus formation, mature zygotic embryo explants were used on basal Murashige and Skoog medium with 9.05 μM 2,4-dichlorophenoxyacetic acid and 3% sucrose. Embryogenic callus was transferred to liquid basal Murashige and Skoog medium with 4.52 μM 2,4-dichlorophenoxyacetic acid and 3% sucrose in order to increase the mass of the embryogenic culture. After pretreatment with potassium citrate, cells were placed into embryo initiation medium with 6 g l-1l-proline and a decreased (10 mM) ammonium concentration. Embryos were matured in 1.89 μM abscisic acid containing half-strength Murashige and Skoog medium and converted into plants bothin vivo andin vitro at up to a 97% efficiency.

Metabolic flux distribution for the optimized production of l-glutamate

Abstract A comprehensive metabolic network was proposed for glutamic acid bacteria and used in a stoichiometrically based flux balance model for l -glutamate production. Theoretical metabolic pathways leading to optimized glutamate overproduction were determined for several specific cell growth rates; variation in the fluxes was obtained. The off-line extracellular analyses throughout the batch fermentation with Brevibacterium flavum showed the accumulation of arginine, aspartate, lysine, alanine, proline, lactate, α-ketoglutarate, succinate, pyruvate, and gluconate in the medium in addition to glutamate. Metabolic flux distributions in the cells throughout the fermentation were determined using the model in combination with the extracellular analyses of the metabolites. The flux distribution maps showed that the cells utilized the TCA cycle in part whereas the glyoxylate bypass was active throughout the fermentation. The results also indicated that the phosphate pentose shunt played an important role in the glutamate fermentation. These diversions in the pathways and certain metabolic reactions depending on the fermentation periods and conditions are also presented in this paper.

Effect of bioreactor configuration on the growth and maturation of Picea sitchensis somatic embryo cultures

Somatic embryo suspension cultures of Picea sitchensis (Sitka spruce) derived from two cell lines, SS03 and SS10, were grown in shake flasks, air-lift, bubble, stirred tank and hanging stirrer bar bioreactors. Cell line SS03 yielded freely suspended and individual stage 1 embryos, while the embryos of SS10 were present in large aggregates. Compared to shake flasks, proliferation in bioreactors resulted in increased biomass; however, cell line morphology influenced the effect of different bioreactor configurations on growth and maturation of embryo cultures. Somatic embryos grown in shake flasks and bioreactors were matured on gelled solid medium and in submerged culture where gelled solid medium was covered with a layer of liquid medium. The number of stage 3 (mature) embryos produced from SS03 in the bubble bioreactor was significantly higher than those from stirred tank and hanging stirrer bar bioreactors with both solid medium and submerged culture. Submerged culture was unsuitable for SS10 embryo maturation.

A KINETIC MODEL FOR ACTINORHODIN PRODUCTION BY STREPTOMYCES COELICOLOR A3(2)

Abstract The fermentation kinetics of an extracellular antibiotic, actinorhodin, by Streptomyces coelicolor were studied in a batch system. A simple model was proposed using the Logistic equation for growth, the Luedeking–Piret equation for actinorhodin production and Luedeking–Piret-like equations for glucose and oxygen consumptions. The model appeared to provide a reasonable description for each parameter during the growth phase.

Characteristics of antibiotic production in a multiphase system

Abstract The production of an extracellular antibiotic, actinorhodin, was investigated in a 2-litre bench-top bioreactor. In order to determine the behaviour of Streptomyces coelicolor cells in a two liquids system, a perfluorocarbon (PFC), which is an oxygen carrier, was added to the fermentation medium. The presence of 50% (v/v) PFC in the fermentation medium resulted in a five-fold increase in the final antibiotic concentration. As a result of the importance of drop size distribution and the liquid-liquid interfacial area in such multiphase systems, the effects of dispersed phase (PFC phase) volume fraction on size distribution and on interfacial area were investigated. The results showed that the size distribution was positively skewed and that the liquid-liquid interfacial area increased with increasing PFC concentration. The liquid-liquid interfacial area was proportional to PFC concentration to the power 1·53.

Somatic embryogenesis of pepper in bioreactors: a study of bioreactor type and oxygen-uptake rates

Somatic embryogenesis of pepper, Capsicum annuum var. Ace, was performed in an airlift bioreactor and a magnetically stirred hanging-stirrer-bar bioreactor, each with 1.81 working volume. All stages of embryogenesis, from growth of embryogenic suspension cultures to embryo maturation, were performed in the bioreactor as a series of drain-and-fill batches, keeping the cells and embryos in the bioreactor all the time. When two bioreactors were compared in terms of percentage embryogenesis and visually observed quality of mixing, under different rates of aeration and stirring, the performance of the magnetically stirred bioreactor was better. The effects of inoculum type and inoculum level on the percentage embryogenesis were also investigated. Under the optimum conditions, embryogenesis was 98%, with 57 embryos/ml. Oxygen-uptake rates of cultures in different stages of embryogenesis were different, the highest being in the embryogenic suspension culture and the lowest during embryo maturation.

Actinorhodin production by Streptomyces coelicolor A3(2): kinetic parameters related to growth, substrate uptake and production

The dynamics of an Streptomyces coelicolor A3(2) culture in a 20-l computer-controlled batch bioreactor was investigated both experimentally and theoretically. In defined medium, depending on the initial conditions, the calculated value of some of the kinetic parameters were: maximum specific growth rate, 0.03 h−1; death rate constant, 1.4–6.3 × 10−3 h−1; observed biomass yield, 0.21 g cells g−1 glucose and the maintenance coefficient for the cells, 0.0448 g glucose g−1 cells h−1. According to both experimental observations and the Luedeking-Piret model, actinorhodin production was found to be growth-associated. This paper provides the first published quantitative information on the main kinetic parameters describing the activity of S. coelicolor in batch culture.

Production of actinorhodin by Streptomyces coelicolor in batch and fed-batch cultures

Abstract Fed-batch fermentations of Streptomyces coelicolor in chemically defined medium in a 20 litre bioreactor were used to produce actinorhodin. Concentrated solutions of phosphate and nitrate with or without glucose were fed intermittently or continuously. Continuous feeding of glucose alone was found to be the best strategy for actinorhodin production. In this case, the specific productivity was almost twice that obtained in conventional batch fermentation.