Perfluorocarbon-mediated aeration applied to recombinant protein production by virus-infected insect cells

Perfluorocarbon (PFC) was used as an oxygen carrier in the cultures of insect cells and virus-infected insect cells. The cell suspensions were placed on a planar layer of PFC, which was re-oxygenated in an outer aeration unit and continuously recirculated, and were agitated by two sets of impeller blades, lower one of which was set in such a way that the ridge of the blade touched the PFC layer. The maximum cell density attained in the PFC-mediated aeration culture was higher than that in surface aeration culture. On viral infection, a recombinant protein yield was significantly high in the PFC-mediated aeration culture as compared with that in the surface aeration culture, though the production was largely decreased by setting apart the lower set of the blade from the PFC-medium interface. These results showed that the PFC-mediated aeration would be a useful technique for insect cell/baculovirus expression system. Overall mass-transfer coefficient K(L) for oxygen was examined in both the PFC-mediated aeration and surface aeration systems, by using a flask whose dimensions were identical to those of spinner flasks used for the cultures. The K(L) value in the PFC-mediated system was 2.60x10(-3)cms(-1), 1.6 times higher than that in the surface aeration system, when impeller blades were positioned at PFC-medium and medium-air interfaces, respectively. However, the K(L) values in both the PFC-mediated and surface aeration systems were decreased and their differences were brought so close, as the blade was set apart from the interfaces. DO behavior in the cultures was well explained by the model calculation using the determined K(L) values and oxygen-consumption rates of viable cells. This calculation further suggested that crucial DO, under which recombinant protein productions were unsuccessful, was 0.24-0.5ppm (3-7%) in the insect cell/baculovirus expression system.     

Cultivation of Arabidopsis cell cultures in a stirred bioreactor at variable oxygen levels: Influence on tocopherol production

Abstract

In plants, an increased production of toxic oxygen species is commonly observed under low oxygen stress, but cellular responses still have to be fully investigated. Plant cell cultures can be a valuable tool to study plant metabolic responses to various environmental stresses including low oxygen condition. Arabidopsis suspension cultures growing in shake flasks were subjected to hypoxia by stopping shaking for different intervals, showing an increase of the antioxidant metabolite α‐tocopherol. In order to obtain a more controlled condition, cultivation of Arabidopsis suspension cultures was established in a 5‐l stirred bioreactor. A constant aeration of 20% dissolved oxygen was found to be the most suitable for cell growth. A 4‐h anoxic shock was induced by suspending the aeration and flushing into the vessel with nitrogen. During the anoxic stress, tocopherol levels resulted increased at the end of the treatment, indicating that the complete oxygen deprivation, indeed, induced a defence response involving antioxidant metabolism. The presence of an oxidative stress as a consequence of anoxic condition was also confirmed by the increased levels of H₂O₂. Overall, these results indicate that Arabidopsis suspension cultures grown in a stirred bioreactor can be a useful in vitro system for investigating low oxygen stress.     

The Aeration of Catharanthus roseus L. G. Don Suspension Cultures in Airlift Bioreactors: The Inhibitory Effect at High Aeration Rates on Culture Growth

An investigation was carried out to examine the effect of aerationon the growth of Catharanthus roseus suspension cultures inairlift bioreactors. A high aeration rate (0·86 v.v.m.)was found to inhibit the growth of cultures. Venting culturesat a high rate with low oxygen content gas mixtures was equallyinhibitory to culture growth, showing that high aeration wasnot inhibitory as a result of oxygen toxicity. The dissolvedcarbon dioxide tension was found to be lower in cultures operatedat high aeration than those operated at low aeration. Supplyingexogenous CO₂ to cultures at high aeration restored the CO₂tension to values normally encountered at a low aeration rate,and was found to alleviate the inhibitory effects at high aeration.However, further increasing the CO₂ supply to cultures was foundto be severely inhibitory to growth. Therefore, the growth ofC. roseus cultures is very sensitive to dissolved CO₂ concentration,growth being inhibited at values either higher or lower thanan optimum. Key words: Aeration, carbon dioxide, Catharanthus roseus suspension culture     

The effect of aeration and metabolic inhibitors on resistance to amphotericin in starved cultures of Candida albicans.

The development of resistance to amphotericin methyl ester, measured in terms of the amount of drug required to induce a standard rate of release of K+ from suspensions of washed organisms, has been followed in Candida albicans in starved cultures under controlled conditions of aeration, stirring and temperature. Resistance develops at a rate which increases with the rate of aeration, limited by the onset of damage due to turbulence. Resistance decreases rapidly if gassing with N2 is substituted for aeration, but sensitivity does not reach that of exponentially growing cells. Resumption of aeration is followed by a slow recovery of resistance. The addition of inhibitors of protein synthesis (trichodermin, verrucarin) or uncoupling agents (2,4-dinitrophenol, sodium azide) at the beginning of starvation results in an increased rate of development of resistance. Adding inhibitors at a later stage, when resistance has developed after 72 h aeration, does not affect the decrease in resistance produced by gassing with N2 but the presence of trichodermin or verrucarin delays the recovery of resistance o     

The tumbling reactor: a new reactor design for biotechnology Cultivation of the phototrophic micro-alga Scenedesmus communis

A new system for the growth of mammalian cells was developed and its biotechnological suitability was proven by the sterile cultivation of the phototrophic micro-alga Scenedesmus communis, as an easy to cultivate model organism. For this purpose, the bioreactor was combined with a newly developed equipment for irradiation of large scale cultures of phototrophic micro-organisms. Cultivation was performed in a 40 liter scale at 25°C and a pH of 6.0 to 6.5. The aeration gas consisted of 2% or 7% of CO2 in N2. The aeration rate was Vgas = 19.6 L.h-1, the stirring speed was n = 70 or 56 min-1. The new reactor consists of 8 totally immersed fluorescent tubes located around a cylinder for bubble free aeration which is moving within the cultivation broth. Results of the cultivation reveal the suitability of this newly designed system for biotechnological use and its high scale-up potential.     

Porphyrin formation byArthrobacter globiformis

The extraction properties, absorption spectra, and chromatographic behaviour of the red extracellular pigment in cultures ofArthrobacter globiformis grown in a mineral salts-glucose medium were identical with those of coprophyrin III. Iron and zinc in small amounts were required for maximum production of the porphyrin. Large amounts of iron failed to suppress porphyrin accumulation in spite of their complete removal from the medium by the bacteria. The effect of iron on porphyrin metabolism in microorganisms is discussed. Accumulation of the porphyrin was highest with moderate aeration; it was almost completely suppressed upon exposure of porphyrin-accumulating cultures to low as well as high aeration rates. At a low aeration rate it was restored by addition of succinic acid, glutamic acid or δ-aminolevulinic acid but at a high aeration by δ-aminolevulinic acid only. At a low aeration rate more porphyrin was formed from δ-aminolevulinic acid than at a high rate.     

Oxygen supply strongly influences metabolic fluxes,the production of poly(3-hydroxybutyrate) and alginate,and the degree of acetylation of alginate in Azotobacter vinelandii

The aim of this study was to evaluate carbon flux in Azotobacter vinelandii using metabolic flux analysis (MFA) under high and low aeration conditions to achieve an improved understanding of how these changes could be related to alginate acetylation and PHB production. Changes in oxygen availability had a considerable impact on the metabolic fluxes and were reflected in the growth rate, the specific glucose consumption rate, and the alginate and PHB yields. The main differences at the metabolic flux level were observed in three important pathways. The first important difference was consistent with respiratory protection; an increase in the flux generated through the tricarboxylic acid (TCA) cycle for cultures grown under high aeration conditions (up to 2.61 times higher) was observed. In the second important difference, the fluxes generated through pyruvate dehydrogenase, phosphoenol pyruvate carboxykinase and pyruvate kinase, all of which are involved in acetyl-CoA metabolism, increased by 10, 43.9 and 17.5%, respectively, in cultures grown under low aeration conditions compared with those grown under high aeration conditions. These changes were related to alginate acetylation, which was 2.6 times higher in the cultures with limited oxygen, and the changes were also related to a drastic increase in PHB production. Finally, the glyoxylate shunt was active under both of the conditions that were tested, and a 2.79-fold increase was observed in cultures that were grown under the low aeration condition.     

Control of growth and differentiation of bioreactor cultures of Physcomitrella by environmental parameters

The effect of physical and chemical environmental parameters on growth and differentiation of suspension cultures of the moss Physcomitrella patens in bioreactors was investigated. By supplementation of the aeration gas with 2% (v/v) CO₂ as well as by continuous illumination, growth of this photoautotrophic growing batch culture was markedly enhanced, resulting in a doubling time of 1.2d. The growth rate of semicontinuously growing bioreactor cultures was not affected by controlling the pH of the culture medium with set points at 4.5 or 7.0. However, growth of the culture at pH 7.0 resulted in increased caulonema development, thus showing a distinct effect on moss differentiation. The impact on research and plant biotechnological applications of the potential to control moss growth and differentiation by environmental parameters is discussed.     

Repeated-batch operation of surface-aerated fermentor for bioethanol production from the hydrolysate of seaweed Sargassum sagamianum

In this study, we investigated the feasibility of sustainable long-term bioethanol production from the hydrolysate of a brown seaweed, Sargassum sagamianum. Because the hydrolysate was prepared as a liquid solution using a hightemperature liquefying system, a repeated-batch operation was utilized as the operational strategy for bioethanol production. Additionally, we used surface aeration to improve bioethanol production from the hydrolysate containing C5 monosaccharides such as xylose. In this study, the C5 monosaccharide-utilizable yeast strain Pichia stipitis was used for bioethanol production. Therefore, based on this repeated-batch flask culture, we designed a surface-aerated repeated-batch fermentor culture, in which the aeration was finely controlled at 100 ml/min and delivered into the headspace of a 2.5-l fermentor. When the medium was replaced every 48 h, bioethanol was continuously produced for 200 h under repeated-batch fermentor culture, where the level of bioethanol production was about 9~10 (g/l). Additionally, the bioethanol yield based on the reducing sugar was about 0.386, which was the average value throughout four consecutive cultures and was about 74.5% of the theoretical value. In addition, the bioethanol yield based on quantitative TLC analyses of glucose and xylose was about 0.431, which was the average value throughout four consecutive cultures and was about 84.3% of theoretical value. Consequently, throughout this repeated-batch operation, we demonstrated that it was actually feasible to produce bioethanol from the hydrolysate of seaweed S. sagamianum. In addition, the approach described here is a practical strategy for commercial bioethanol production from seaweed, particularly for finely controlling aeration through surface aeration.     

Role of mitochondria in the sex-directed flocculation of a fission yeast

Cultures of Schizosaccharomyces pombe NCYC 132, a homothallic haplont, were grown anaerobically to stationary phase and then aerated. Cells flocculated within 1 hr of aeration. Competence for flocculation induction decayed as the cultures were allowed to age in stationary phase. Heat-killed cells were not inducible, but flocculated if induced before they were killed. However, massive ultraviolet irradiation, which resulted in the inability of the cells to form colonies when plated, did not stop induction. Added at the time of aeration, cyanide, azide, and dinitrophenol inhibited induction. So did membrane-specific drugs, such as nystatin and polymyxin, as well as inhibitors of protein synthesis such as cycloheximide, puromycin, and neomycin. Chloramphenicol, at saturating concentration, had no effect on flocculation induction when added at the time of aeration. But added to aerobically growing cells long before the last generation, chloramphenicol completely inhibited floc formation. The results indicate that induction of competence to form flocs requires mitochondrial function and cytoplasmic protein synthesis.     

Experimental study of a ceramic microsparging aeration system in a pilot-scale animal cell culture

The oxygen supply of cell cultures with the aid of free gas bubbles is an efficient process strategy in pharmaceutical production. If the cell-damaging impact of gas bubbles is reduced, direct aeration becomes a practical solution with scale-up potential and comparatively high oxygen transfer rates. In this paper a microsparging aeration system made of porous ceramic was compared with bubble-free membrane aeration. The sparging system was used for the long-term cultivation of mammalian cells in 2- to 100-L scale bioreactors and produced bubble sizes of 100-500 microm in diameter. Using a scale of 2.5 and 30 L, a cell density of 2.6 x 10(6) cells/mL was attained. When a 100-L scale was used, a density of 1.1 x 10(6) cells/mL was achieved, whereas a comparable membrane-aerated system showed a cell density of 2.2 x 10(6) cells/mL. At relatively low agitation rates of less than 70 rpm in the sparged bioreactors, a homogeneous and constant oxygen concentration was kept in the medium. As a result of the different foam-forming tendency caused by the lower gas flow of the ceramic sparger compared to that of the standard aeration systems, we were able to develop an appropriate process control strategy. Furthermore, oxygen transfer measurements for the common stainless steel sparger and the ceramic sparger showed a 3-fold higher oxygen transfer coefficient for the ceramic sparger.     

Outdoor cultivation ofArthrospira platensis during autumn and winter in temperate climates

Arthrospira (Spirulina) platensis M2 was grown outdoors in 50-mm diameter tubular reactors under the climatic conditions of central Italy (Florence) from September to December 1995 and in March 1996. Except for September, the cultures temperature was regulated. Mean productivities of 0.83, 0.44 and 0.61 g dry wt L^–1 d^–1 were achieved in autumn (September–October), winter (November–December) and March, respectively. In autumn and winter, the photosynthetic efficiency of the cultures and the degree of correlation between productivity and solar irradiance were significantly greater than in summer. The effect of cell density and aeration rate on productivity was evaluated in September. The productivity of cultures operated at high supra-optimal population density was about 30% less at high aeration rate (1.0 LL^–1 min^–1), and 50% less at standard aeration rate (0.17 LL^–1 min^–1), than that of control cultures kept at optimal population density and standard aeration rate. The reduction of productivity in high-density cultures was due to lower daylight output rates and higher night biomass losses (the latter were particularly relevant under standard aeration conditions). The main factor limiting productivity in closed reactors during autumn was the night temperature. Heating the cultures during daylight hours on sunny days did not cause any significant increase of the yields, since under sunlight the unheated cultures also reached the optimal temperature for growth early in the morning. On cloudy days, the day-time temperature of the unheated cultures remained well below the optimum, however this had only a limited effect on productivity since algal growth was mainly light-limited.     

Redox driven metabolic tuning: carbon source and aeration affect synthesis of poly(3-hydroxybutyrate) in Escherichia coli

Growth and polymer synthesis were studied in a recombinant E. coli strain carrying phaBAC and phaP of Azotobacter sp. strain FA8 using different carbon sources and oxygen availability conditions. The results obtained with glucose or glycerol were completely different, demonstrating that the metabolic routes leading to the synthesis of the polymer when using glycerol do not respond to environmental conditions such as oxygen availability in the same way as they do when other substrates, such as glucose, are used. When cells were grown in a bioreactor using glucose the amount of polymer accumulated at low aeration was reduced by half when compared to high aeration, while glycerol cultures produced at low aeration almost twice the amount of polymer synthesized at the higher aeration condition. The synthesis of other metabolic products, such as ethanol, lactate, formate and acetate, were also affected by both the carbon source used and aeration conditions. In glucose cultures, lactate and formate production increased in low agitation compared to high agitation, while poly(3-hydroxybutyrate) synthesis decreased. In glycerol cultures, the amount of acids produced also increased when agitation was lowered, but carbon flow was mostly redirected towards ethanol and poly(3-hydroxybutyrate). These results indicated that carbon partitioning differed depending on both carbon source and oxygen availability, and that aeration conditions had different effects on the synthesis of the polymer and other metabolic products when glucose or glycerol were used.     

The uptake of copper by cell suspension cultures of Phaseolus vulgaris L. cv. SAXA

In experiments with cell suspension cultures of Phaseolus vulgaris L. cv. SAXA it could be demonstrated that gas-shocks caused by aeration decrease the uptake of copper. An aeration period of 30 h prior to the addition of copper was found to be sufficient to reestablish the copper uptake system. EDTA/sucrose solutions seemed to be adequate to remove the adsorbed copper. The time course of the uptake of copper showed saturation behaviour, whereas the velocity of the uptake was dependent on the given concentration of copper.     

动植物细胞或组织生物反应器悬浮培养过程中的通气方式

通气在动植物细胞或组织生物反应器培养过程中起着至关重要的作用,而同时通气过程所产生的机械损伤力亦可对细胞造成直接的伤害,因此,通气方式是动植物细胞或组织生物反应器培养过程设计与工程放大的关键技术之一。本文综述了动植物细胞或组织生物反应器悬浮培养过程中三种主要通气(异养培养时又称供氧)方式的结构特点,及其对气液传质、生物量、代谢产物量和细胞损伤的影响,以及改进的新型通气方式和几种通气方式的融合并用。     

Influence of aeration on hydrogen cyanide biosynthesis byPseudomonas aeruginosa

Batch cultures ofPseudomonas aeruginosa were able to produce only low levels of cyanide during logarithmic growth with adequate aeration. The reduction of aeration caused a rapid increase in the ability of such cultures to produce hydrogen cyanide. The immediacy and the magnitude of this response depended on the oxygen level, with a concentration of 4% in the aeration gas giving optimal results. The reestablishment of normal aeration resulted in a cessation of the increase of the culture's cyanogenic capacity. This effect appeared to be a combination of inactivation of the hydrogen cyanide synthase and repression of synthesis of this enzyme.     

Mathematics for the Majority

The development of an experimental system which can be used for demonstrating population studies in Chlorella vulgaris, and which is appropriate for use in secondary schools, is described. A culture medium was prepared on the basis of established inorganic nutrient requirements, and several cultures were set up to test the effects of variations in nutrient composition, temperature, pH, light and aeration. A satisfactory system was eventually found which produced an ideal (sigmoidal) population-growth curve within three weeks. A similar pattern of results, though with different growth rates and maximum population levels, was obtained both for controlled and ambient conditions of light and temperature, and for aerated tube and non-aerated flask cultures. Non-aerated tube cultures were relatively ineffective. Two methods were used, the counting of cells and the measurement of optical density, for estimating population density, and comparative results were obtained for serial dilutions of a standard culture.     

Formation of 5,10-Dihydrophenazine from Phenazine by Pseudomonas cepacia IFO 15124 at Low Oxygen Tensions

5,10-Dihydrophenazine (H₂Phen) was formed from phenazine (Phen) by Pseudomonas cepacia IFO 15124 in growing cultures at low oxygen tensions. Effects of culture conditions on microbial reduction of Phen with this strain were investigated. Under optimized conditions, the transformation of Phen to H₂Phen by this strain gave the molar conversion yield of 30%. However, H₂Phen was not detected in the culture medium when the strain was incubated with Phen with sufficient aeration.     

The effects of aeration on glucose catabolism in Penicillium expansum.

Polyacrylamide-disc gel electrophoresis and quantitative enzyme assays showed that the pathways of glucose catabolism and secondary metabolism in Penicillium expansum were dependent on the degree of aeration of the cultures. The isoenzyme patterns and specific activities of aldolase and succinate dehydrogenase indicated that glycolysis and the tricarboxylic acid cycle operated under conditions of both limited and efficient aeration (i.e. in cultures grown statically or on an orbital shaker). At high levels of aeration the growth rate was faster and synthesis of extracellular pectolytic enzymes was enhanced, whilst the activities of glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase showed that the pentose-phosphate shunt was important in glucose catabolism during the trophophase of growth. In contrast, under conditions of low aeration this latter pathway was virtually undetectable, growth was slower, pectolytic enzyme production low and large concentrations of secondary metabolites (6-methylsalicylic acid, patulin and citrinin) accumulated.     

Physiological and morphological characteristics of stationary phase vibrio cells able to support phase growth

Growth of phase alpha 3a on stationary phase Vibrio cultures requires micro-aerophilic conditions and is inhibited by aeration. Since pre-conditioning of the bacteria by allowing them to stand for 24 h after shaking for 3 d is an important aspect of the stationary phase phage growth system, various physiological and morphological characteristics of the stationary phase cells during the transition from shaking to standing were investigated. Shaken stationary phase cells were less viable and more sensitive to ultraviolet irradiation and heat than standing stationary phase cells. During pre-conditioning the small, non-flagellated cells present in shaken stationary phase cultures underwent morphological changes and became large, flagellated rods which resembled exponential phase cells. The transition of stationary phase cells from shaking to standing was associated with a marked increase in total RNA synthesis but a rapid and large decrease in total protein synthesis. Intracellular concentrations of ATP in shaken stationary phase cells were 53% lower than those in standing stationary phase cells. Studies on leucine uptake indicated that its transport was inhibited by isoleucine and that the major part (90%) of the total leucine uptake was due to a shared system for uptake of both amino acids. Shaken stationary phase cells transported less leucine than standing stationary phase cells. Inhibition of phage growth in aerated stationary phase cultures was not due to the prevention of phase absorption by shaking. It is suggested that the observed differences between shaken and standing stationary phase cells could be due to aeration affecting the template specificity of the Vibrio RNA polymerase.