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A Dense Cell Culture System for Aerobic Microorganisms Using a Shaken Ceramic Membrane Flask with Surface Aeration KAMOSHITA YUYA, OHASHI RYO, SUZUKI TAKAHIRO | |
| Journal of fermentation and bioengineering, Vol.85, No.2, 1998, pp. 218-222 | |
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Aeration of the head space in a shaken ceramic membrane flask (SCM flask) capped with 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 230rpm 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. | |
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A dense cell culture system for microorganisms using a shake flask incorporating a porous ceramic filter SUZUKI T. ; KAMOSHITA Y. ; OHASHI R. | |
| Journal of fermentation and bioengineering, 1997, vol. 84, no2, pp. 133-137 | |
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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 235g/l at 220h 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. | |
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A fully automated robotic system for high throughput fermentation Zimmermann HF, Rieth J. | |
| Clinics in Laboratory Medicine (2007) 27(1):209-214 | |
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High throughput robotic systems have been used since the 1990s to carry out biochemical assays in microtiter plates. However, before the application of such systems in industrial fermentation process development, some important specific demands should be taken into account. These are sufficient oxygen supply, optimal growth temperature, minimized sample evaporation, avoidance of contaminations, and simple but reliable process monitoring. A fully automated solution where all these aspects have been taken into account is presented. | |
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A new scale-up method based on the effect of ventilation on aerated fermentation systems Tanaka, H.; Ishikawa, H.; Nobayashi H.; Takagi, Y. | |
| Journal of Fermentation and Bioengineering, Volume 72, Issue 3, 1991, Pages 204-209 | |
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A new scale-up method based on the effect of ventilation on aerated fermentation systems was developed. The volatilization rate constant (kv) of the volatile compound was proposed as an index for showing quantitatively the effect of ventilation on the aerated fermentation systems. The kv value of a volatile compound 2-octanol, an inhibitor of cell growth, in jar fermentor was affected mainly by the air flow rate and to an extent by the agitation speed. In a scale-up of culture systems having a volatile growth inhibitor as a rate limiting factor, the need to consider both the oxygen transfer rate and the ventilation effect was demonstrated in a simple yeast model culture containing 2-octanol. It was shown quantitatively that an Erlenmeyer flask with a cotton plug had essentially no ventilation effect when compared to a jar fermentor. An aerated-flask having a forced-air system was therefore designed and a new method of scale-up from this flask to jar fermentor on the basis of the kv value was proposed. The usefulness of both the aerated-flask and this new scale-up method was demonstrated with the model yeast culture. | |
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A new wireless system for decentralised measurement of physiological parameters from shake flasks Vasala A, Panula J, Bollók M, Illmann L, Haelsig C, Neubauer P | |
| Microb Cell Fact., 2006; 5:8 | |
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BACKGROUND: Shake flasks are widely used because of their low price and simple handling. Many researcher are, however, not aware of the physiological consequences of oxygen limitation and substrate overflow metabolism that occur in shake flasks. Availability of a wireless measuring system brings the possibilities for quality control and design of cultivation conditions. RESULTS: Here we present a new wireless solution for the measurement of pH and oxygen from shake flasks with standard sensors, which allows data transmission over a distance of more than 100 metres in laboratory environments. This new system was applied to monitoring of cultivation conditions in shake flasks. The at-time monitoring of the growth conditions became possible by simple means. Here we demonstrate that with typical protocols E. coli shake flask cultures run into severe oxygen limitation and the medium is strongly acidified. Additionally the strength of the new system is demonstrated by continuous monitoring of the oxygen level in methanol-fed Pichia pastoris shake flask cultures, which allows the optimisation of substrate feeding for preventing starvation or methanol overfeed. 40 % higher cell density was obtained by preventing starvation phases which occur in standard shake flask protocols by adding methanol when the respiration activity decreased in the cultures. CONCLUSION: The here introduced wireless system can read parallel sensor data over long distances from shake flasks that are under vigorous shaking in cultivation rooms or closed incubators. The presented technology allows centralised monitoring of decentralised targets. It is useful for the monitoring of pH and dissolved oxygen in shake flask cultures. It is not limited to standard sensors, but can be easily adopted to new types of sensors and measurement places (e.g., new sensor points in large-scale bioreactors). | |
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A novel method for the measurement of oxygen mass transfer rates in small-scale vessels ORTIZ-OCHOA Kenny, DOIG Steven D., WARD John M., BAGANZ Frank | |
| Biochemical engineering journal (2005) Vol. 25, No. 1, 63-68 | |
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A novel method for the measurement of the volumetric oxygen transfer coefficient, kLa, using the catechol-2,3-dioxygenase (XylE) bio-oxidation of catechol yielding 2-hydroxymuconic semialdehyde (2-HS), has been developed for small-scale systems. This method was kinetically characterized and validated by comparison to other established techniques. The bio-oxidation rate was found to be zero order over a catechol concentration from 2 to 7 mM l-1. When the enzyme concentration was in excess and the bio-oxidation rate was mass transfer limited, indicated by a percent DOT of zero, the oxygen transfer rate was determined from the linear increase in product concentration. The method was validated in a 21 stirred tank vessel equipped with a DOT probe and connected to a gas mass spectrometer. The novel method yielded similar kLa values when compared to the dynamic gassing out method under the same conditions. The applicability of this novel method for small-scale devices was demonstrated by measuring kLa values of up to 150 h-1 in shaken microplates with a working volume of only 200 μl. | |
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A novel parallel shaken bioreactor system for continuous operation Akguen, Ali; Maier, Bernd; Preis, Diana; Roth, Birthe; Klingelhoefer, Renata; Buechs, Jochen | |
| Biotechnology progress. - 20 (2004), 6, S. 1718-1724 | |
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A novel continuous bioreactor system was developed as a shaken culture vessel for the investigation of the growth kinetics and product formation of microorganisms in milliscale. The novel bioreactor system mainly consists of a specially designed 250-mL shake flask with two inlets, one for gas supply and one for medium supply, and one combined outlet on the side of flask for exhaust gas and culture liquid. As a result of the circulating motion of the fermentation broth in the shake flask, the maximum liquid height reaches the edge of the outlet and the fermentation broth is accelerated into the outlet by centrifugal force. Additionally, the excess fermentation broth leaving the culture vessel is continuously driven by the exhaust gas. Because of the small scale and the simple handling it is possible to operate many of these shaken bioreactor vessels simultaneously. By using parallel vessels operated at different dilution rates on the same shaker, the data for a complete biomass over dilution rate (X-D) diagram of a biological culture can be evaluated in an efficient manner, thus saving money, materials, and time. Continuous fermentations of the yeast Saccharomyces cerevisiae H1022 (ATCC 32167) in the shaken bioreactor system and in a conventional stirred tank fermentor showed very similar results. | |
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A remedy to oxygen limitation problem in antibiotic production: addition of perfluorocarbon Murat Elibol, Ferda Mavituna | |
| Biochemical Engineering Journal, Volume 3, Issue 1, 1 February 1999, Pages 1-7 | |
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n this study, the effect of an oxygen carrier, perfluorocarbon, on actinorhodin fermentation by Streptomyces coelicolor A3(2) was investigated using a chemically defined medium in 2 and 20 l bioreactors. The inclusion of 50% (v/v) perfluorocarbon in the fermentation medium resulted in a five-fold increase in the maximum antibiotic concentration. The use of perfluorocarbon also caused remarkable increases in both glucose and oxygen consumption rates. Moreover, the increasing concentrations of perfluorocarbon improved the dissolved oxygen profile by raising the minimum dissolved oxygen concentration. It was found that observed increases in the antibiotic production were linearly related to the volumetric oxygen uptake rates. This result could perhaps be attributed to the enhancement of oxygen transfer in S. coelicolor cultures due to the higher oxygen solubilities of the fermentation medium through inclusion of perfluorodecalin. | |
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A simple and inexpensive method for investigating microbiological, enzymatic, or inorganic catalysis using standard histology and microbiology laboratory equipment: assembly, mass transfer properties, hydrodynamic conditions and evaluation Seletzky J.M., Otten K., Lotter S., Fricke J., Peter C.P., Maier H.R., Buechs J. | |
| Biotechnic and Histochemistry 81(4-6), 133-138, 2006. | |
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We introduce a generic, simple, and inexpensive method for performing microbiological, enzymatic, or inorganic catalysis with solids using standard histology and microbiology laboratory equipment. Histology cassettes were used to standardize hydrodynamic conditions and to protect the catalysts and their solid supports. Histology cassettes have the following advantages: they are readily available, inexpensive, solvent and acid resistant, automatable, and the slots in the cassette walls allow liquid to circulate freely. Standard Erlenmeyer flasks were used as reaction vessels. We developed a new camera to observe the movement and position of the histology cassettes as well as the liquid in the Erlenmeyer flasks. The camera produces a stable image of the rotating liquid in the Erlenmeyer flask. This visualization method revealed that in a 250 ml Erlenmeyer flask, stable operating conditions are achieved at a shaking frequency of 300 rpm and a fill volume of 30 ml. In vessels with vertical walls, such as beakers or laboratory bottles, the movement of the histology cassette is not reproducible. Mass transfer characterization using a biological model system and the chemical sulfite-oxidation method revealed that the histology cassette does not influence gas-liquid mass transfer. | |
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A study of oxygen transfer in shake flasks using a non-invasive oxygen sensor Gupta A, Rao G | |
| Biotechnol Bioeng. 2003 Nov 5;84(3):351-8 | |
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We describe a study of oxygen transfer in shake flasks using a non-invasive optical sensor. This study investigates the effect of different plugs, presence of baffles, and the type of media on the dissolved oxygen profiles during Escherichia coli fermentation. We measured the volumetric mass transfer coefficient (k(L)a) under various conditions and also the resistances of the various plugs. Finally, we compared shake flask k(L)a with that from a stirred tank fermentor. By matching k(L)a's we were able to obtain similar growth and recombinant protein product formation kinetics in both a fermentor and a shake flask. These results provide a quantitative comparison of fermentations in a shake flask vs. a bench-scale fermentor and should be valuable in guiding scale-up efforts. Copyright 2003 Wiley Periodicals, Inc. | |
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Achieving shaker flask-scale cell densities in miniprep-scale cultures with baffled culture tubes and growth medium H15 VIGIL L. J.; DANIELSON P. B. ; SOLLARS C. ; YEE T. M. ; FOGLEMAN J. C. | |
| BioTechniques (2000) Vol. 29, No. 6, pp 1207-1209 | |
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In conclusion, modifications to the preparation of H15 broth, including the preparation of glucose-free medium and filter sterilization, significantly improve the storage and reliability of H15 growth medium. Furthermore, the use of baffled glass culture tubes enhances aeration and, thus, the performance of H15 broth in 2-mL overnight cultures now supports growth to the same high cell densities that previously were only attainable using much larger and more expensive baffled flasks. These modifications offer researchers the opportunity to significantly boost the efficiency and yield of the small-scale bacterial cultures that are used for a wide variety of applications including the preparation of plasmid DNA and the heterologous expression of proteins. | |
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Advances in understanding and modeling the gas-liquid mass transfer in shake flasks Maier U., Losen M., Buechs J. | |
| Biochemical Engineering Journal 17, 155-167, 2004 | |
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The gas–liquid mass transfer in 250 ml shake flasks has previously been sucessfully modelled on basis of Higbie’s penetration theory. The current contribution presents advances in understanding and modelling the gas–liquid mass transfer in shake flasks at waterlike liquid viscosity in flask sizes between 50 and 1000 ml. An experimental investigation of the maximum gas–liquid mass transfer capacity OTRmax using the sodium sulphite system was extended to relative filling volumes of 4–16%, shaking diameters of 1.25, 2.5, 5, 7, 10 cm and shaking frequencies of 50–500 rpm for the above flask sizes. Simultaneously, the previous model of the gas–liquid mass transfer was extended to a “two sub-reactor model” to account for different mechanisms of mass transfer in the liquid film on the flask wall and the bulk of the liquid rotating within the flask. The shake flask is for the first time considered to be a two-reactor system consisting of a stirred tank reactor (bulk liquid) and a film reactor (film on flask wall and base). The mass transfer into the film on the flask wall and base at “in-phase” operating conditions is described by Higbie’s penetration theory. Two different mass transfer theories were applied to successfully describe the mass transfer into the bulk liquid: a model by Kawase and Moo-Young and a model by Gnielinski. The agreement between the new modelling approach, which requires absolutely no fitting parameters and the experimental is within ±30%. The applicability of the models to a biological system was shown using a Pichia pastoris culture. This is particularly notable since geometrically non-similar liquid distributions in very different sizes of shaking flasks are covered. A comparable description of the gas–liquid mass transfer in bubble aerated reactors like stirred tanks is absolutely out of reach. A spatially- and time-resolved consideration of the mass transfer in the liquid film on the flask wall and base has shown that the validity of Higbie’s theory sensitively depends on the film thickness and contact time. | |
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Aeration in the Laboratory Lockhart WR, Squires RW | |
| Adv Appl Microbiol. 1963;5:157-187. | |
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review article regarding aeration in shake flask and fermenters The aeration of cultures often seems a difficult field which the microbiologist tries to avoid. Finding apparently conflicting advice in the literature, he may decide to ignore aeration altogether rather than becorne involved. It may be useful to consider the factors which have given rise to this state of affairs. The effects on biological systems of oxygen availability, and of oxidation-reduction potentials in general, are complex and not well understood. Problems encountered in delivering oxygen to cultures involve technical and sometimes obscure concepts in physical chemistry and engineering. The investigator must choose among a great variety of equipment and techniques for carrying out aeration. Finally, the measurernent of aeration effciency is not easy. Although we have a number of analytical techniques, each of them is subject to severe limitations. | |
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AERATION REQUIREMENTS FOR THE GROWTH OF AEROBIC MICROORGANISMS Charles G. Smith and Marvin J. Johnson | |
| J Bacteriol. 1954 September; 68(3): 346–350. | |
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With Serratia marcescens the per cent yield of cells based on substrate utilized, the total cell concentration, and the live cell count hae been shown to vary directly with aeration efficiency. | |
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Ajmalicine production by cell cultures of Catharanthus roseus: from shake flask to bioreactor Hens J. G. ten Hoopen, Walter M. van Gulik, Jurriaan E. Schlatmann, Paulo R. H. Moreno, J. L. Vinke, J. J. Heijnen, Robert Verpoorte | |
| Plant Cell, Tissue and Organ Culture, Volume 38, Numbers 2-3, 1994 | |
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The productivity of a cell culture for the production of a secondary metabolite is defined by three factors: specific growth rate, specific product formation rate, and biomass concentration during production. The effect of scaling-up from shake flask to bioreactor on growth and production and the effect of increasing the biomass concentration were investigated for the production of ajmalicine by Catharanthus roseus cell suspensions. Growth of biomass was not affected by the type of culture vessel. Growth, carbohydrate storage, glucose and oxygen consumption, and the carbon dioxide production could be predicted rather well by a structured model with the internal phosphate and the external glucose concentration as the controlling factors. The production of ajmalicine on production medium in a shake flask was not reproduced in a bioreactor. The production could be restored by creating a gas regime in the bioreactor comparable to that in a shake flask. Increasing the biomass concentration both in a shake flask and in a stirred fermenter decreased the ajmalicine production rate. This effect could be removed partly by controlling the oxygen concentration in the more dense culture at 85% air saturation. | |
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An experimental comparison of respiration measuring techniques in fermenters and shake flask: exhaust gas analyzer vs. RAMOS device vs. respirometer Seletzky J.M, Noack U., Hahn S., Knoll A., Amoabediny G., Buechs J. | |
| Journal of Industrial Microbiology & Biotechnology 34(2), 123-130, 2007 | |
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Respiration measurement is applied as a universal tool to determine the activity of biological systems. The measurement techniques are difficult to compare, due to the vast variety of devices and analytical procedures commonly in use. They are used in fields as different as microbiology, gene engineering, toxicology, and industrial process monitoring to observe the physiological activity of living systems in environments as diverse as fermenters, shake flasks, lakes and sewage plants. A method is introduced to determine accuracy, quantitation limit, range and precision of different respiration measurement devices. Corynebacterium glutamicum cultures were used to compare an exhaust gas analyzer (EGA), a RAMOS device (respiration measurement in shake flasks) and a respirometer. With all measuring devices it was possible to determine the general culture characteristics. The EGA and the RAMOS device produced almost identical results. The scatter of the respirometer was noticeably higher. The EGA is the technique of choice, if the reaction volume is high or a short reaction time is required. The possibility to monitor cultures simultaneously makes the RAMOS device an indispensable tool for media and strain development. If online monitoring is not compulsive, the respiration of the investigated microbial system extremely low, or the sample size small, a respirometer is recommended. | |
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An inexpensive system to provide sparged aeration to shake flask cultures Robert S. Donovan, Campbell W. Robinson, Bernard R. Glick | |
| Biotechnology Techniques, Volume 9, Number 9 / September 1995, pp. 665-670 | |
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We have constructed a durable and inexpensive aeration system for providing sparged aeration of bacterial cultures grown in shake flasks. A detailed description of the apparatus is presented, sufficient to allow other researchers to construct such a system. | |
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Analysis of Pellet Formation of Aspergillus niger Based on Shear Stress FUJITA MASANORI, IWAHORI KEISUKE, TATSUTA SIGERU, YAMAKAWA KOICHIRO | |
| Journal of fermentation and bioengineering, Vol.78, No.5 (1994) pp. 368-373 | |
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A theoretical discussion on pellet formation of Aspergillus niger based on shear rate or averge velocity gradient (G value) was made for both cases of a reactor with paddle and a rotationally shaken flask. The average diameter of pellets (d) and the number of pellets formed per unit volume (n) are linearly to the G value below 400 1/s on a logarithmic plot, and a regression equaton, n=(1.37×10^<-5>)・d^<-3.25>, was obtained regardless of the agitation and inoculation conditions. Under non-turbulent agitaiton conditions, the average number of spores forming a single pellet was found to be approximately proportional to the two-thirds power of the concentration of the spore inculated. These exponents are very close to the theoretical one. Consequently it is concluded that G value is a representative index for the evaluation of pellent formation capability, verifying the validity of the proposed theory. | |
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Apparatus for monitoring the oxygen uptake and carbon dioxide production of fermentations A. L. Jensen, J. S. Schultz | |
| Biotechnology and Bioengineering, Volume 8, Issue 4 , Pages 539 - 548 (1966) | |
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The requirements of the continuous analysis of effluent gas streams from aerated flash and tank fermentors are described, as are instrumental devices for measuring the oxygen and carbon dioxide content of fermentor gases. The use of a specially designed sequential gas sample for monitoring four fermentations simultaneously and a system for precise control of low air flow and pressure is explained. Equations for calculating carbon dioxide production or oxygen consumption rates and respiratory quotients are given. A discussion of the operating characteristics of a device for automatic translation of aeration data between fermentors is presented. | |
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Application of response surface methodology for glucosyltransferase production and conversion of sucrose into isomaltulose using free Erwinia sp. cells Haroldo Yukio Kawaguti, Eiric Manrich , Hélia Harumi Sato | |
| Electronic Journal of Biotechnology ISSN: 0717-3458, Vol. 9 No. 5, Issue of October 15, 2006 | |
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Isomaltulose is a structural isomer of sucrose commercially used in food industries. Glucosyltransferase produced by Erwinia sp. D12 catalyses an intramolecular transglucosylation of sucrose giving isomaltulose. The Experimental design and response surface methodology were applied for the optimization of the nutrient concentration in the culture medium for the enzyme production in shaken flasks at 200 rpm and 30ºC. The three variables involved in this study were sugar cane molasses, bacteriological peptone and yeast extract Prodex Lac SD®. The statistical analysis of the results showed that, in the range studied, all the factors had a significant effect (p < 0.05) on glucosyltransferase production and the highest enzyme activity was observed in culture medium containing sugar cane molasses (160 g/L), bacteriological peptone (20 g/L) and yeast extract Prodex Lac SD® (15 g/L). Maximum glucosyltransferase activity of 29.88 U/mL was achieved in a 6.6-L fermenter using the optimized medium. Free Erwinia sp. D12 cells were used for isomaltulose production from sucrose during fifteen successive batches. The final isomaltulose concentration of 75.6% obtained in the first batch increased to 77.21% (mean value) in the other fourteen batches and the productivity of 1.1 g/L x hr was obtained in batch process. | |
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Applied shaking technology / ed. by Jochen Buechs Buechs J. | |
| Elsevier. 2004. - S. 155 - 223. : graph. Darst... - (Biochemial engineering journal ; 17,3 : Special issue) | |
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Special Edition: Applied Shaking Technology Applied Shaking Technology. International Symposium No2, Tarritown, New York | |
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Bacterial leaching of a sulfide ore by Thiobacillus ferrooxidans and Thiobacillus thiooxidans: I. Shake flask studies Hector M. Lizama, Isamu Suzuki | |
| Biotechnology and Bioengineering, 1988, Volume 32, Issue 1, Pages 110 - 116 | |
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Bacterial leaching of a sulfide ore containing pyrite, chalcopyrite, and sphalerite was studied in shake flask experiments using Thiobacillus ferrooxidans and Thiobacillus thiooxidans strains isolated from mine sites. The Fe2+grown T. ferrooxidans isolates solubilized sphalerite preferentially over chalcopyrite leaching 7-10% Cu, 68-76% Zn, and 10-22% Fe from the ore in 18 days. The sulfur grown T. thiooxidans isolates leached Zn much more slowly and very little Fe, with a Cu-Zn extraction ratio twice the value obtained with T. ferrooxidans. The ore adapted T. ferrooxidans started solubilizing Cu and Zn without a lag period. The ore-adapted T. thiooxidans extracted Cu as well as T. ferrooxidans, but the extraction of Zn or Fe was still much slower in the low-phosphate medium, while in the high-phosphate medium it approached the value obtained with T. ferrooxidans. A high Cu-Zn extraction ratio of 0.34 was obtained with T. thiooxidans in the low phosphate medium. In the mixed-culture experiments with T. ferrooxidans and T. thiooxidans, the culture behaved as T. thiooxidans in the low-phosphate medium with a higher Cu-Zn extraction ratio and as T. ferrooxidans in the high-phosphate medium with a lower Cu-Zn extraction ratio. It is concluded that T. ferrooxidans and T. thiooxidans solubilize sulfide minerals by different mechanisms. | |
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Batch culture of bacteria under controlled gaseous conditions C. J. Efthymiou, M. Veit | |
| Applied Microbiology and Biotechnology, Volume 7, Number 2 ,1979, pp 121-128 | |
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A flask, designed for direct gassing of batch cultures of bacteria, was evaluated for its use in studying oxygen absorption rates (OAR) and suitability for physiological studies under various controlled atmospheres. Such flasks, aerated directly without shaking, yielded an OAR (up to 1.2 mmol O2/l/min) that was comparable to or higher than those obtained in conventional flasks aerated by shaking. Direct aeration in combination with shaking resulted in OAR values that were elevated and most favorable for growth of oxygen demanding bacteria (ap5 mmol O2/l/min). In comparison with controls, the direct method of aeration in combination with shaking proved most efficient and least dependent on the surface to volume ratio of the aerated solution. In experiments with the facultative anaerobe Streptococcus faecalis 10Cl, grown in controlled aerobic, anaerobic, and mixed gas (CO2-free air, air-plus-CO2, N2-plus-CO2) environments, a specific anaerobic requirement for CO2 could be established. The wide range of gaseous environments possible renders the newly tested flask useful for comparative biochemical studies, especially when the gaseous condition of culture is a factor of critical importance. | |
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Beta-glucan production by Botryosphaeria rhodina in different bench-top bioreactors Selbmann L; Crognale S; Petruccioli M | |
| Journal of Applied Microbiology, Volume 96, Number 5, May 2004 , pp. 1074-1081(8) | |
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Evaluation of the technical feasibility of transferring bgr-glucan production by Botryosphaeria rhodina DABAC-P82 from shaken flasks to bench-top bioreactors. Methods and Results: Three different bioreactors were used: 3 l stirred tank reactor (STR-1) equipped with two different six-blade turbines; STR as above but equipped with a three-blade marine propeller plus draft-tube (STR-2); 2 l air-lift column reactor (ALR) equipped with an external loop. STR-1, tested at three different stirrer speeds (300, 500 and 700 rev min-1) appeared to be less suitable for bgr-glucan production by the fungus, being maximum production (19·4 g l-1), productivity (0·42 g l-1 h-1) and yield (0·48 g g-1 of glucose consumed) markedly lower than those obtained in shaken culture (29·7 g l-1, 1·23 g l-1 h-1 and 0·61 g g-1, respectively). Better performances were obtained with both STR-2 and ALR. With the latter, in particular, the increase of production was accompanied by reduced fermentation time (25·7 g l-1 after only 22 h); productivity and yield were highest (1·17 g l-1 h-1 and 0·62 g g-1 of glucose consumed, respectively). Conclusion: Using an air-lift reactor with external loop, the scaling up from shaken flasks to bench-top bioreactor of the bgr-glucan production by B. rhodina DABAC-P82 is technically feasible. Significance and Impact of the Study: Although culture conditions are still to be optimized, the results obtained using the ARL are highly promising. | |
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Biodegradation of the herbicide glyphosate by filamentous fungi in platform shaker and batch bioreactor Castro JV Jr, Peralba MC, Ayub MA | |
| J Environ Sci Health B. 2007; 42(8): 883-886 | |
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The biodegradation conducted by microorganisms on herbicide glyphosate (N-phosphonomethylglycine) was investigated. Five strains of filamentous fungi belonging to the Fusarium genre were grown on Czapeck medium without phosphorous and supplemented with the addition of glyphosate. The assays were conducted to determine the ability of use as a phosphorous source, the inhibition caused by presence of herbicide, and the biodegradation in shaker and bioreactor by Fusarium strains. It was observed that the herbicide did not show any negative effect on microrganisms by quantity of the biomass. Among the strains tested, no inhibition was noted by the addition of glyphosate even at a high concentration. All strains studied were able to biodegrade it and use the herbicide as a phosphorous source. The formation of consortium was not better than the strains tested in pure culture. The biodegradation in the bioreactor was better than in the shaker. However, there wasn't any influence on biodegradation rate by changing the amount of oxygen in the system. | |
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Bioreactor considerations for secondary metabolite production from plant cell tissue culture: indole alkaloids from Catharanthus roseus PAYNE G. F. ; PAYNE N. N. ; SHULER M. ; | |
| Biotechnology and bioengineering 1988, vol. 31, no9, pp. 905-912 | |
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Batch shake flask studies with Catharanthus roseus demonstrated that alkaloid production commenced only after growth had slowed or ceased. To obtain high alkaloid productivities for extended periods, a hormone-free production medium was used. To develop a readily scalable process, both immobilized and suspended cell systems were studied. In the immobilized cell systems, growth, glucose utilization, and alkaloid production were suppressed; for the case of membrane entrapped cells this suppression was observed to be reversible. Based on the oxygen requirements of the cells, and the oxygen transfer capabilities of a pneumatically agitated bubble column, conditions were established that allowed the growth and production dynamics observed in shake flasks to be reproduced in the air sparged column reactor. The requirement for the aseptic exchange of growth for production medium was satisfied by using a coarse cotton filter and coupling filtration to aeration. With this filtration system, media can be rapidly and completely exchanged and the filter can be quickly and effectively backwashed. By coupling filtration to aeration, a two-stage batch operation can be employed while requiring only a single bioreactor. Studies with this system demonstrated its capabilities for alkaloid production. | |
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Bioreactor processes based on disposable materials for the production of recombinant proteins from mammalian cells Matthieu Stettler | |
| Thèse EPFL, no 3947 (2007). Dir.: Florian Wurm | |
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The objective of industrial cell culture technology is to produce high value-added therapeutic proteins that are well suited for the treatment of infectious diseases, cancer, and autoimmune diseases. Nowadays, an important focus is the reduction of the time and costs from the discovery of the candidate molecule to the full-scale production. Relying on enabling technologies is critical for meeting this objective. Simultaneously, novel technologies are expected to increase the clinical and commercial success rates. In recent years, new concepts based on innovative disposable materials emerged and appeared to be promising alternatives to standard bioprocessing equipment. This thesis work was focused on the use of such disposable compounds in mammalian cell culture applications, particularly for the containment of the cells, and was aimed at demonstrating their benefit in terms of cost-effectiveness, ease-of-use and flexibility. First, a novel disposable packed cell volume tube was shown to be ideal for the quick and reliable assessment of biomasses. A characterization and validation of the measurement system demonstrated that it could replace time-consuming and less accurate cell counting methods. Then, to determine the appropriate growth conditions for cells in culture, small-scale single-use tubes were orbitally agitated with conventional lab shakers. This approach was found to be well-suited for multi-parameter optimization strategies. A systematic characterization of the liquid mass transfer in shake tubes proved that sufficient oxygen was available even at cell densities beyond 1 ×107 cells mL-1. Non-invasive optical methods were used to assess the dissolved oxygen variations in various orbital shake vessels, from the milliliter to the multi-liter scale. This simple and yet powerful cultivation principle, orbital shaking, was scaled-up to pilot and production scales. To match the requirements in terms of oxygen transfer at larger scales, a given airflow was provided to replace the gas in the headspace. When required, the airflow was enriched with oxygen. At scales above 20 L, sterile disposable cell cultivation bags were used to contain the cells. Combining orbital shake technology and disposable cell culture bags with a cylindrical shape was promising, both in terms of efficiency and ease-of-use. Prototype shake bioreactors of 200 L and 1'500 L were designed, constructed and operated with maximal cell densities up to 5-7 ×106 cells mL-1 in batch cultivations of CHO cells. The benefits in terms of time and cost savings were even more obvious when novel shake bioreactors were compared with standard stirred-tank bioreactors. This was shown in a realistic optimization, scale-up and production sequence. Most importantly, this work established that orbital shake technology, unlike other disposable cell cultivation systems, can be used for a wide range of operating scales, from only a few milliliters for optimization purposes up to production scale. | |
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Broth rheology, growth and metabolite production of Beta vulgaris suspension culture: a comparative study between cultures grown in shake flasks and in a stirred tank Rodrguez-Monroy M.; Galindo E. | |
| Enzyme and Microbial Technology, Volume 24, Number 10, 1 July 1999 , pp. 687-693 | |
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Cells of Beta vulgaris have the ability to grow in a stirred tank under an impeller tip speed as high as 95.3 cm seg−1. Comparing this system with cultures performing in shake flasks, a decrease of the cell concentration, betalains production, and growth rate was observed. However, the kinetic profiles of aggregates size and cellular viability were practically the same. The cultures carried out in the fermentor showed a major accumulation of extracellular arabinogalactoprotein and polysaccharide, which is an indication of the cell response to hydrodynamic stress. These extracellular molecules produced a considerable change in the rheology of cell-free medium. This change in the rheology can be playing an important role in the reduction of the actual hydrodynamic stress during cultivation. | |
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Calculating Liquid Distribution in Shake Flasks on Rotary Shakers at Waterlike Viscosities Buechs J., Maier U., Lotter St., Peter C.P. | |
| Biochemical Engineering Science 34(3), 200–208, 2007. | |
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Screening projects in biotechnological industry performed in shake flasks risk unwanted development if not failure, when operating conditions are not suitable. Limited knowledge, however, is available for the mechanistical design of operating conditions in this type of bioreactor. The fundamental engineering variables are influenced by the geometry of the rotating bulk liquid: for momentum transfer, the contact area between the liquid and the flask inner wall is the friction area, and for mass transfer, the wetted wall exposed to the surrounding air is the mass exchange area. To assess the geometry of the rotating bulk liquid moving inside a shaken Erlenmeyer flask, with respect to the mentioned important engineering variables mentioned, a mechanistical model for the liquid distribution in shake flasks is described in this work. The model is based on a superposition of two individual movements: a circular translatoric movement and a rotation of the flask counteracting the first motion to keep the shake flasks’ spatial orientation. If the effect of viscosity is neglected, the liquid distribution results in an exactly symmetrical paraboloid. A comparison of the calculated liquid distribution with photographs shows very good qualitative agreement of the real liquid distributions by the model equations. Quantitative agreement has been demonstrated by comparison of the liquid height. Furthermore, model equations are presented for the calculation of the contact area between the liquid and the flask wall. This may eventually lead to a prediction of the volumetric power consumption. Similarly, the calculation of the mass transfer area (i.e. liquid surface area and wetted flask wall) is presented. | |
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Changes in alginate molecular mass distributions, broth viscosity and morphology of Azotobacter vinelandii cultured in shake flasks PENA C., CAMPOS N., GALINDO E. | |
| Applied microbiology and biotechnology (1997), vol. 48, no4, pp. 510-515 | |
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The effect of different aeration conditions during the culture of Azotobacter vinelandii on the production and molecular mass of alginate was evaluated in shake flasks. In baffled flasks, the bacteria grew faster and produced less alginate (1.5 g/1) than in conventional (unbaffled) flasks (4.5 g/1). The viscosity of the culture broth was also influenced by the type of flask. Higher final viscosities were attained in unbaffled flasks [520 cP (520 mPa s)] as compared to baffled flasks (30 cP). This latter phenomenon was closely related to the changes in the molecular mass distribution. In either cases, the mean molecular mass increased with culture age; however, at the end of the fermentation, the mean molecular mass of the alginate obtained in unbaffled flasks was fivefold higher than that obtained in baffled flasks. As the culture proceeded, the cells of Azotobacter grown in unbaffled flasks increased in diameter, whereas those cultured in baffled flasks decreased in size. | |
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Characterisation of operation conditions and online monitoring of physiological culture parameters in shaken 24-well microtiter plates. Kensy F, John GT, Hofmann B, Buechs J | |
| Bioprocess Biosyst Eng. 2005 Nov;28(2):75-81 | |
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A new online monitoring technique to measure the physiological parameters, dissolved oxygen (DO) and pH of microbial cultures in continuously shaken 24-well microtiter plates (MTP) is introduced. The new technology is based on immobilised fluorophores at the bottom of standard 24-well MTPs. The sensor MTP is installed in a sensor dish reader, which can be fixed on an orbital shaker. This approach allows real online measurements of physiological parameters during continuous shaking of cultures without interrupting mixing and mass transfer like currently available technologies do. The oxygen transfer conditions at one constant shaking frequency (250 1/min) and diameter (25 mm) was examined with the chemical sulphite oxidation method. Varied filling volumes (600-1,200 microL) of Escherichia coli cultures demonstrated the importance of sufficient oxygen transfer to the culture. Cultures with higher filling volumes were subjected to an oxygen limitation, which influenced the cell metabolism and prolongated the cultivation time. The effects could be clearly monitored by online DO and pH measurements. A further study of different media in an E. coli fermentation elucidated the different growth behaviour in response to the medium composition. The MTP fermentations correlated very well with parallel fermentations in shake flasks. The new technique gives valuable new insights into biological processes at a very small scale, thus enabling parallel experimentation and shorter development times in bioprocessing. | |
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Characterisation of the gas-liquid mass transfer in shaking bioreactors Maier U, Buechs J. | |
| Biochem Eng J. 2001 Mar;7(2):99-106 | |
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The maximum gas-liquid mass transfer capacity of 250ml shaking flasks on orbital shaking machines has been experimentally investigated using the sulphite oxidation method under variation of the shaking frequency, shaking diameter, filling volume and viscosity of the medium. The distribution of the liquid within the flask has been modelled by the intersection between the rotational hyperboloid of the liquid and the inner wall of the shaking flask. This model allows for the calculation of the specific exchange area (a), the mass transfer coefficient (k(L)) and the maximum oxygen transfer capacity (OTR(max)) for given operating conditions and requires no fitting parameters. The model agrees well with the experimental results. It was furthermore shown that the liquid film on the flask wall contributes significantly to the specific mass transfer area (a) and to the oxygen transfer rate (OTR). | |
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Characterization and application of an optical sensor for quantification of dissolved O2 in shake-flasks Wittmann C, Kim HM, John G, Heinzle E | |
| Biotechnol Lett. 2003 Mar;25(5):377-80 | |
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On-line measurement of dissolved O2 in shake-flasks was realized via immobilized sensor spots containing a fluorophore with an O2-dependent luminescent decay time. An unaffected sensor signal during 80 autoclaving cycles suggests multi-usage of sensor equipped shake-flasks. The sensor had a response time of 6 s. Quantification of gas-liquid mass transfer revealed maximum kLa values of 150 h(-1), from which maximum O2 transfer capacity of 33 mM h(-1) was calculated. Liquid volume and shaking frequency have a strong influence on kLa. Exemplified by cultivations of Corynebacterium glutamicum the importance of shaking rate for O2 supply of bacterial cultures is shown. Sampling of microbial cultures with intermittent shaking of a few minutes can cause O2 limitation. Based on the results of this work a simple and straightforward tool is now available for accurate O2 sensing in shake-flasks, which are widely used in microbial cultivations. | |
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Characterization of Agitation Effects in Shaken Flasks RICHARD P. RHODES and ELMER L. GADEN, Jr. | |
| Ind. Eng. Chem., 1957, Vol. 49, No. 8, 1233-1236 | |
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Though shaken flasks are commonly used in process studies, too little is known about performance. Experiments with rotary and reciprocating units provide needed data. | |
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Characterization of bimodal cell death of insect cells in a rotating-wall vessel and shaker flask Cowger NL, O'Connor KC, Hammond TG, Lacks DJ, Navar GL | |
| Biotechnol Bioeng. 1999; 64(1):14-26 | |
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In previous publications, we reported the benefits of a high-aspect rotating-wall vessel (HARV) over conventional bioreactors for insect-cell cultivation in terms of reduced medium requirements and enhanced longevity. To more fully understand the effects that HARV cultivation has on longevity, the present study characterizes the mode and kinetics of Spodoptera frugiperda cell death in this quiescent environment relative to a shaker-flask control. Data from flow cytometry and fluorescence microscopy show a greater accumulation of apoptotic cells in the HARV culture, by a factor of at least 2 at the end of the cultivation period. We present a kinetic model of growth and bimodal cell death. The model is unique for including both apoptosis and necrosis, and further, transition steps within the two pathways. Kinetic constants reveal that total cell death is reduced in the HARV and the accumulation of apoptotic cells in this vessel results from reduced depletion by lysis and secondary necrosis. The ratio of early apoptotic to necrotic cell formation is found independent of cultivation conditions. In the model, apoptosis is only well represented by an integral term, which may indicate its dependence on accumulation of some factor over time; in contrast, necrosis is adequately represented with a first-order term. Cell-cycle analysis shows the percent of tetraploid cells gradually decreases during cultivation in both vessels. For example, between 90% and 70% viability, tetraploid cells in the HARV drop from 43 +/- 1% to 24 +/- 4%. The data suggests the tetraploid phase as the likely origin for apoptosis in our cultures. Possible mechanisms for these changes in bimodal cell death are discussed, including hydrodynamic forces, cell-cell interactions, waste accumulation, and mass transport. These studies may benefit insect-cell cultivation by increasing our understanding of cell death in culture and providing a means for further enhancing culture longevity. | |
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Characterization of Gas-Liquid Mass Transfer Phenomena in Microtiter Plates Hermann R., Lehmann M., Buechs, J. | |
| Biotechnology and Bioengineering, Vol. 81, No. 2, January 20, 2003 | |
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Gas-liquid mass transfer properties of shaken 96-well microtiter plates were characterized using a recently described method. The maximum oxygen transfer capacity (OTRmax), the specific mass transfer area (a), and the mass transfer coefficient (kL) in a single well were determined at different shaking intensities (different shaking frequencies and shaking diameters at constant filling volume) and different filling volumes by means of sulfite oxidation as a chemical model system. The shape (round and square cross-sections) and the size (up to 2 mL maximum filling volume) of a microtiter plate well were also considered as influencing parameters. To get an indication of the hydrodynamic behavior of the liquid phase in a well, images were taken during shaking and the liquid height derived as a characteristic parameter. The investigations revealed that the OTRmax is predominantly dependent on the specific mass transfer area (a) for the considered conditions in round-shaped wells. The mass transfer coefficient (kL) in round-shaped wells remains at a nearly constant value of about 0.2 m/h for all shaking intensities, thus within the range reported in the literature for surface-aerated bioreactors. The OTRmax in round-shaped wells is strongly influenced by the interfacial tension, determined by the surface tension of the medium used and the surface properties of the well material. Up to a specific shaking intensity the liquid surface in the wells remains horizontal and no liquid movement can be observed. This critical shaking intensity must be exceeded to overcome the surface tension and, thus, to increase the liquid height and enlarge the specific mass transfer area. This behavior is solely specific to microtiter plates and has not yet been observed for larger shaking bioreactors such as shaking flasks. In square-shaped microtiter plate wells the corners act as baffles and cause a significant increase of OTRmax, a, and kL. An OTRmax of up to 0.15 mol/L/h can be reached in square-shaped wells. | |
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Characterization of mixing in shaker table containers GARDNER J. ; TATTERSON G. | |
| Biotechnology and bioengineering (1992), vol. 39, no7, pp. 794-797 | |
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Mixing in shaker table beakers is studied using dye dispersion to measure mixing times. Experimental conditions range from the laminar regime into the turbulent mixing. Different flow patterns occurring in the beakers are reported for the mixing. The transition Reynolds number is determined. Rotational speed of the table, volume of material to be mixed, and viscosity of the material are studied as to their effects on mixing time. A graphical mixing time correlation is provided which is useful for the translation of mixing from laboratory scale to pilot scale | |
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Characterization of oxygen transfer conditions and their effects on Phaffia rhodozyma growth and carotenoid production in shake-flask cultures Yuan-Shuai Liu, Jian-Yong Wu and Kwok-ping Ho | |
| Biochemical Engineering Journal, Volume 27, Issue 3, January 2006, Pages 331-335 | |
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This study characterizes the effects of oxygen transfer on the growth and carotenoid (astaxanthin) production of the red yeast Phaffia rhodozyma in liquid cultures. The yeast culture on a liquid medium at 20 °C had a critical dissolved oxygen (DO) concentration of 10% air-saturation (0.8 mg/L) and a maximum specific oxygen uptake rate of 60 mg O2/g cell h. In 250-mL shake-flasks with 50 ml liquid medium, poor oxygen transfer resulted in constantly lower DO level than the critical DO in the culture. Both the biomass and carotenoid yields increased with the decrease in liquid volume in flasks and the increase in shaker speed, e.g., 8.0 g dw/L biomass and 7.4 mg/L carotenoid with 50 mL liquid at 200 rpm versus 10.7 g dw/L biomass and 15.5 mg/L carotenoid with 20 mL liquid at 250 rpm. The oxygen transfer coefficient (KLa) was correlated to shaker speed (N) and liquid volume (VL) by KLa = 0.141 N0.88 (VL/Vo)−0.80 based on linear regression of experimental data (R2 = 0.997, Vo = flask volume). The carotenoid yield showed a strong linear correlation with the oxygen transfer rate (R2 = 0.989). These results show that oxygen supply is crucial for carotenoid production in P. rhodozyma liquid cultures. | |
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Cheese whey-induced high-cell-density production of recombinant proteins in Escherichia coli Mikko I Viitanen, Antti Vasala, Peter Neubauer, Tapani Alatossava | |
| Microbial Cell Factories 2003, 2:2 | |
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Background: Use of lactose-rich concentrates from dairy processes for the induction of recombinant gene's expression has not received much attention although they are interesting low cost substrates for production of recombinant enzymes. Applicability of dairy waste for induction of recombinant genes in Escherichia coli was studied. Clones expressing Lactobacillus phage muramidase and Lactobacillus alcohol dehydrogenase were used for the experiments. Results: Shake flask cultivations in mineral salt medium showed that cheese whey or deproteinised whey induced gene expression as efficiently as IPTG (isopropyl-β-D-thiogalactopyranoside) or pure lactose. Addition of yeast extract or proteolytically degraded whey proteins did not improve the recombinant protein yield. In contrast, addition of yeast extract to the well-balanced mineral salt medium decreased the product yield. Feeding with glycerol provided sufficient amount of easily assimilable carbon source during the induction period without preventing lactose intake and induction by lactose. High-cell-density fed-batch cultivations showed that product yields comparable to IPTG-induction can be achieved by feeding bacteria with a mixture of glycerol and concentrated whey permeate during the induction. Conclusion: Whey and concentrated whey permeate can be applied as an alternative inducer in recombinant high-cell-density fed-batch fermentations. The yield of the recombinant product was comparable to fermentations induced by IPTG. In low-cell-density shake flask experiments the yield was higher with whey or whey permeate than with IPTG. | |
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Closure effects on oxygen transfer and aerobic growth in shake flasks NIKAKHTARI H ; HILL GA | |
| Biotechnology and bioengineering, 2006, vol. 95, no1, pp. 15-21 | |
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Oxygen mass transfer in shake flasks is an important aspect limiting the culture of aerobic micro-organisms. In this work, mass transfer of oxygen through a closure and headspace of shake flasks is investigated. New equations for prediction of kGa in shake flasks with closures are introduced. Using Pseudomonas putida, microbial growth on glucose (fast metabolism) and phenol (slow metabolism) in shake flasks with closures were studied, considering both substrate and oxygen restrictions. A combined model for oxygen mass transfer and microbial growth is shown to accurately predict experimental oxygen concentrations and oxygen yield factors during growth experiments more accurately than previous models. | |
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Coffee filters as shake-flask closures Paul A. Hartman, Michael B. Roof | |
| Biotechnology Techniques, Volume 1, Number 2, 1987, pp 143-144 | |
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Coffee filters are suitable for use as inexpensive and convenient shake-flask closures for propaqating bacteria under aerobic conditions. Gaseous exchange rates are excellent. Sterility is maintained when two layers of filter are used. | |
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Combination of On-line pH and Oxygen Transfer Rate Measurement in Shake Flasks by Fiber Optical Technique and Respiration Activity MOnitoring System (RAMOS) Marco Scheidle, Johannes Klinger and Jochen Buechs | |
| Sensors 2007, 7, 3472-3480 | |
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Shake flasks are commonly used for process development in biotechnology industry. For this purpose a lot of information is required from the growth conditions during the fermentation experiments. Therefore, Anderlei et al. developed the RAMOS technology [1, 2], which proviedes on-line oxygen and carbondioxide transfer rates in shake flasks. Besides oxygen consumption, the pH in the medium also plays an important role for the successful cultivation of micro-organisms and for process development. For online pH measurement fiber optical methods based on fluorophores are available. Here a combination of the on-line Oxygen Transfer Rate (OTR) measurements in the RAMOS device with an on-line, fiber optical pH measurement is presented. To demonstrate the application of the combined measurement techniques, Escherichia coli cultivations were performed and online pH measurements were compared with off-line samples. The combination of on-line OTR and pH measurements gives a lot of information about the cultivation and, therefore, it is a powerful technique for monitoring shake flask experiments as well as for process development. | |
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Comparative applications of bioreactor and shake-flask systems in the laboratory treatment of oily sludge Okpokwasili GC, Oton NS | |
| Intern. Journ. of Environment and Waste Management, Volume 1, Number 1, 2006 , pp. 49-60 | |
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This study investigates the remediation efficiencies of a locally fabricated bioreactor system and a shake-flask system, using samples of oily sludge with varying concentrations of NPK fertiliser. Hydrocarbon removal efficiency of 46% and 24.2% was recorded for the stirred slurries containing 15- and 0-g NPK, respectively, in the bioreactor; whereas values of 40.1%, 33.2% and 18.9%, respectively, were obtained for the shaken flasks containing 0.3-, 0.113- and 0-g NPK at the end of this study. Corresponding values of 26% and 11.8% were obtained for the unstirred slurries; as well as 21.9%, 23.1% and 14.3% for the unshaken flasks. Nutrient amendment was shown to enhance the rate of oil removal in the following order: 0.2% NPK > 0.075% NPK > no NPK. However, the combined effects of nutrient addition and agitation proved to be the most effective treatments as confirmed by gas chromatography. pH and temperature values obtained were generally in optimal range (6.5 8.5 and 29 31°C, respectively). This preliminary investigation shows the potential utilisation of NPK in large-scale bioremediation of soils contaminated by oily sludge, using a well-stirred bioreactor system. | |
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COMPARATIVE EVALUATION OF CULTURE TUBE CLOSURES Ladd FC | |
| Can. J. Microbiol. 9(5): 727–729 (1963) | |
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[...] the literature contains no comaprison of the efficacy of any closures, except in parallel with conventional cotton plugs. The need for a comparative evaluation of culture vessel closures is apparent. This communication reports results of a preliminary examination of a variety of commercially available culture tube closures. [...] | |
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Comparison of torque method and temperature method for determination of power consumption in disposable shaken bioreactors Raval K., Kato Y, Buechs J. | |
| Biochemical Engineering Journal 34(3), 224–227, 2007. | |
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Disposable bioreactors are increasingly used in bioprocess development of animal cell cultures. However, the engineering parameters of the present disposable bioreactors are very difficult to characterise. Characterisation of any bioreactor is important to evaluate the suitability of operating conditions and absolutely necessary for successful scale-up. In this research work, an attempt is made to validate a characterisation method which is simple and can be used for any given volume of disposable bioreactors larger than 2 L. Two evaluation methods, namely the temperature method and the well established torque method were used for determination of power consumption in 2 L and 20 L disposable shaken bioreactors. The trend of the values of power consumption was shown with respect to shaking frequency and filling volume. Results indicate that quite reasonable values of power consumption can be obtained by the temperature method and the torque method, which are in good agreement with each other (error <30%). | |
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Complete suspension of solid particles in a shaking vessel Kato Y, Hiraoka S, Tada Y, Shirota T, Koh S-T, Lee Y-S, Yamaguchi T | |
| Kagaku Kogaku Ronbunshu, 1995, Vol. 21, pp 948-952 (in japanese) | |
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The critical circulating frequency and the power consumption for complete suspension of particles, based on the definition of Zwietering, were measured in a shaking vessel containing a solid-liquid system. The critical frequency was correlated by the equation from Baldi's particle model modified with the physical properties of the particle. The power consumption at the critical suspension condition in the shaking vessel was less than that in an agitated vessel with impeller. | |
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Computational-fluid-dynamics (CFD) analysis of mixing and gas–liquid mass transfer in shake flasks Zhang H, Williams-Dalson W, Keshavarz-Moore E, Shamlou PA | |
| Biotechnol. Appl. Biochem. (2005) 41, (1–8) | |
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CFD (computational fluid dynamics) techniques were used to predict mixing and gas-liquid mass transfer in a 250 ml shake flask operating over a range of shaking frequencies between 100 and 300 rev./min, shaking diameters between 20 and 60 mm, and fill volumes between 25 and 100 ml. Interfacial area, a, volumetric mass-transfer coeffcient, kLa, and the power input per unit volume, ev, of the liquid were predicted to be 300 | |
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Continuous Monitoring of Oxygen Concentrations in Air-Purged Shake-Flask Fermentations Phillip H. Hodson | |
| Appl Microbiol. 1970 March; 19(3): 551–554 | |
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Continuous monitoring of the oxygen concentrations in the gas and liquid phases in air-purged shake-flask fermentations was accomplished by means of steam-sterilizable oxygen probes. | |
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Cotton Closure as an Aeration Barrier in Shaken Flask Fermentations
Jerome S. Schultz | |
| Appl Microbiol. 1964 July; 12(4): 305–310. | |
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Oxygen transfer through a cotton plug follows Fick's law of diffusion. The apparent diffusion constant decreases as the density of cotton packing is increased. Formulas are derived which relate oxygen transfer to cotton-plug dimensions. Calculations indicate that the dimensions of a cotton plug can affect both the maximal oxygen uptake rate and the gas composition inside a shaken flask. In some situations, as with large flasks, the cotton closure can become the limiting factor to aeration. | |
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Cultivation of Aspergillus awamori on wheat bran: Ultrastructural description of growth and localization of xylanase by immunogold labeling Adolph S.; Muller S.; Siedenberg D.; Jager K.; Lehmann H.; Schugerl K.1; Giuseppin M. | |
| Journal of Biotechnology, Volume 46, Number 3, 15 May 1996 , pp. 221-234(14) | |
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Aspergillus awamori was culivated in shake flask cultures and in stirred tank reactors on synthetic medium and on complex medium. (with wheat bran substrate), respectively, and samples were taken at different cultivation times. Teh filametous mycelia and pellets of the fungus were analyzed using four different techniques. transmission electron micorscopy (TEM), electron spectroscopic imaging (ESI), electron energy loss spectroscopy (EELS) and immunocytochemical investigation. The ultrastructure of A. awamori and the colonization and decomposition of the wheat bran by the fungus were determined as a function of the cultivation time. The xylanase concentration in the cytoplasm and cell wall of the fungus was high during the fist phase of the cultivation and it gradually decreased due to its stecretion. At the same time the xylanase concentration increased on the surface of the solid substrate as well as in the cultivation medium. The variation of the local distribution of the enzyme xylanase during the cultivation was determined as well. It is shown how the fungus penetrates and decomposes the wheat bran. Phosphorous and sulfur inclusion, determined with ESI and EELS, were found in the vacuoles. | |
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Culture conditions promoting dispersed growth and biphasic production of actinorhodin in shaken cultures of Streptomyces coelicolor A3(2) Doull JL, Vining LC | |
| FEMS Microbiol Lett. 1989; 53(3): 265-268 | |
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Media and culture conditions were developed for experiments on the physiology of secondary metabolism in Streptomyces coelicolor A3(2). Well dispersed mycelial growth was obtained in a buffered starch-glutamate-salts medium; a high (5%) starch concentration and addition of glass beads aided dispersal. Under the conditions developed, production of actinorhodin was suppressed during trophophase growth and began abruptly near the growth maximum. | |
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Culture in the rotating-wall vessel affects recombinant protein production capability of two insect cell lines in different manners Saarinen, Mark; Murhammer, David | |
| In Vitro Cellular & Developmental Biology - Animal, Volume 36, Number 6, June 2000 , pp. 362-366 | |
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The production of recombinant secreted alkaline phosphatase protein in virally infected insect cells was studied in shaker flask and high aspect rotating-wall vessel (HARV) culture. Two commonly used cell lines, Spodoptera frugiperda Sf-9 (Sf-9) and a nonaggregating isolate of the Trichoplusia ni BTI-Tn-5B1-4 (Tn-5B1-4) cell line, Trichoplusia ni Tn-5B1-4-NA (TN-5B1-4-NA), were used and monitored for 120-h postinfection. Different responses to culture in the HARV were seen in the two cell lines. While the Sf-9 cell line was able to produce slightly greater amounts of recombinant protein in the HARV than in shaker flask controls, the Tn-5B1-4-NA cell line produced significantly lesser amounts in the HARV than in the shaker flasks. Both cell lines exhibited longer life spans and longer periods of protein production in HARV culture than in shaker flask culture, presumably due to lower levels of shear encountered in the HARV. The important difference was in the protein production rate responses of the two cell lines. While the protein production rates of Sf-9 cells were comparable in both HARV and shaker flask cultures, the protein production rates of Tn-5B1-4-NA cells were much lower in HARV culture than in shaker flask cultures. The conclusion is drawn that cell line-specific adaptation to the HARV strongly influences recombinant protein production. | |
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Determination of carbon dioxide production rates for mammalian cells in 24-well plates Yang Y, Balcarcel RR | |
| Biotechniques. 2004 Feb;36(2):286-90, 292, 294-5 | |
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In this report, we describe a method for the quantitative determination of carbon dioxide production rates of mammalian cells. Custom-made, reusable, optically clear plugs are used to seal the wells of a 24-well plate. These plugs prevent the loss of CO2 produced by the mammalian cells cultured in bicarbonate-free medium. Measurements of pH, total liquid phase CO2, and viable cell density are used to estimate the average CO2 production rate during a 6-h incubation period. Using this method, four chemicals well-characterized in regards to toxicity, 2,4-dinitrophenol, antimycin A, rotenone, and cyanide, were found to elicit significant changes in CO2 production for given concentrations within 6 h, without inducing a decline in culture viability. Over longer exposure times, similar concentrations caused growth inhibition but not cell death. An assay based on metabolic change corresponding to growth inhibition that is more sensitive than traditional measures of cell death is a feasible complement to existing methods in drug discovery and toxicity testing. | |
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Development of a Novel Box-Shaped Shake Flask with Efficient Gas Exchange Capacity Kato I.; Tanaka H. | |
| Journal of Fermentation and Bioengineering, Volume 85, Number 4, 1998 , pp. 404-409(6) | |
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A novel box-shaped shake flask with membrane filters fitted in its upper sides was developed for efficient gas exchange. During rotation, the liquid flow inside the flask is broken up at the four angles of the flask, while membrane filters in opposite sides serve alternately as gas inlet and outlet ports, depending on the direction in which the flask is moving. Consequently, the oxygen transfer coefficients both across the gas-liquid interface and through the membrane filters are more than ten times higher than those in an Erlenmeyer flask with a cotton plug. The volumetric oxygen transfer coefficient, volatilization rate constant, and cultivation performance of representative strains of microorganisms were used to evaluate the gas exchange capacity of the newly developed shake flask. It was confirmed that the gas exchange capacity of the flask was the same as that of a jar fermentor. | |
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Development of a shaking bioreactor system for animal cell cultures Liu C, Hong L | |
| Biochem Eng J. 2001 Mar;7(2):121-125. | |
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The feasibility of using shake flasks to culture animal cells was evaluated using various sizes of cylindrical shaped vessels as bioreactors. It was found that conditions can be optimized so that hybridoma, Chinese Hamster Ovary cells, and insect cells can be efficiently cultured in the shaking reactors to cell densities comparable to that obtained with stirred-jar bioreactors, and the system is scalable to larger volumes for the production of recombinant proteins or cell mass production in the laboratory | |
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Development of scale-down techniques for investigation of recombinant Escherichia coli fermentations: acid metabolites in shake flasks and stirred bioreactors. Dahlgren ME, Powell AL, Greasham RL, George HA | |
| Biotechnol Prog. 1993 Nov-Dec;9(6):580-586 | |
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We have developed shake-flask screening conditions that are predictive of specific expression of the chimeric toxin, TGF alpha-PE40, by recombinant Escherichia coli JM109 in stirred bioreactors. When a nutrient-rich stirred bioreactor medium was used in shake flasks, neither the extent of growth nor the specific level of recombinant protein expression duplicated the performance in stirred bioreactor fermentations. Incomplete oxidation of glucose and concomitant accumulation of organic acid metabolites, as well as oxygen limitation and lack of pH control, were examined as contributors to the poorer performance in the flask. The medium buffering capacity, initial glucose level, and flask aeration were evaluated to establish the limits of "scale-down" conditions for expression both in a complex nutrient medium (M101) similar to that used in stirred bioreactors and in a defined (FM) medium. Acid metabolites and ethanol were measured as indicators of carbon flow from glucose as well as indirect indicators of oxygen limitation. For the complex M101 medium, optimal shake-flask performance in 250-mL, nonbaffled flasks at 37 degrees C occurred with 0.3 x medium strength, supplementation with 0.3 m HEPES buffer (pH 7.5), and 10 mL of medium per flask. Cultures grown under these conditions produced a maximum density of 3.6 g of dry cell weight/L (as estimated by absorbance measurements at 600 nm) and maintained a pH near neutrality. Additionally, metabolite markers of anaerobic or microaerobic conditions, such as ethanol, lactate, and pyruvate, were not detected, and specific expression of TGF alpha-PE40 was comparable to stirred bioreactors induced for expression at various biomass levels. When culture parameters were controlled within these limits, similar results were also observed in the defined FM medium. | |
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Development, parallelization, and automation of a gas-inducing milliliter-scale bioreactor for high-throughput bioprocess design (HTBD) PUSKEILER R., KAUFMANN K., WEUSTER-BOTZ D. | |
| Biotechnology and bioengineering (2005) vol. 89, no5, pp. 512-523 | |
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A novel milliliter-scale bioreactor equipped with a gas-inducing impeller was developed with oxygen transfer coefficients as high as in laboratory and industrial stirred-tank bioreactors. The bioreactor reaches oxygen transfer coefficients of >0.4 s-1. Oxygen transfer coefficients of >0.2 s-1 can be maintained over a range of 8- to 12-mL reaction volume. A reaction block with integrated heat exchangers was developed for 48-mL-scale bioreactors. The block can be closed with a single gas cover spreading sterile process gas from a central inlet into the headspace of all bioreactors. The gas cover simultaneously acts as a sterile barrier, making the reaction block a stand-alone device that represents an alternative to 48 parallel-operated shake flasks on a much smaller footprint. Process control software was developed to control a liquid-handling system for automated sampling, titration of pH, substrate feeding, and a microtiter plate reader for automated atline pH and atline optical density analytics. The liquid-handling parameters for titration agent, feeding solution, and cell samples were optimized to increase data quality. A simple proportional pH-control algorithm and intermittent titration of pH enabled Escherichia coli growth to a dry cell weight of 20.5 g L-1 in fed-batch cultivation with air aeration. Growth of E. coli at the milliliter scale (10 mL) was shown to be equivalent to laboratory scale (3 L) with regard to growth rate, μ, and biomass yield, Yxs. | |
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Device for sterile online measurement of the oxygen transfer rate in shaking flasks Anderlei T, Buechs J | |
| Biochem Eng J. 2001 Mar;7(2):157-162 | |
|
The oxygen transfer rate (OTR) is the most suitable measurable parameter to quantify the physiological state of a culture of aerobic microorganisms since most metabolic activities depend on oxygen consumption. Online measurement of the oxygen transfer rate in stirred bioreactors is state of the art although technically difficult. However, the online determination of the oxygen transfer rate in shaking bioreactors under sterile conditions has not been possible until recently. A newly developed measuring device eliminates this deficit. Extremely useful information about cultivating conditions and the physiological state of microorganisms can be gained in early stages of research and bioprocess development from many reactors operated in parallel. | |
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Different shake flask closures alter gas phase composition and ajmalicine production in Catharanthus roseus cell suspensions Carolyn W. T. Lee, Michael L. Shuler | |
| Biotechnology Techniques, Volume 5, Number 3 / Mai 1991, pp 173-178 | |
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The type of closure chosen for plant cell cultures can significantly alter the headspace gas composition of a culture, leading to major differences in the production of secondary metabolites. In cell suspension cultures of Catharanthus roseus, ethylene accumulated in cultures with limited gas exchange and appeared to inhibit the production of ajmalicine. The variability in product yields between replicates can also be attributed to gas composition differences. | |
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Disposable shaker flasks E. A. Falch, C.-G. Hedén | |
| Biotechnology and Bioengineering, Volume 5, Issue 3 , Pages 211 - 220 (1963) | |
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A disposable plastic tetrahedron bag is described which is suitable for holding a 50 ml. culture on a shaker table. Bags can be made out of polypropylene or Teflon and they are quite simple and inexpensive. The tetrahedrons make excellent use of the shaker table space and they give a threefold increase in aeration efficiency over Erlenmeyer flasks. | |
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Effect of Agitation on Ligninase Activity and Ligninase Production by Phanerochaete chrysosporium Rajagopalan Venkatadri, Robert L. Irvine | |
| Appl Environ Microbiol. 1990; 56(9): 2684–2691. | |
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The white rot fungus Phanerochaete chrysosporium produces extracellular ligninases as part of its idiophasic ligninolytic system. Agitation has been widely reported to suppress both ligninase production and lignin degradation. Results show that mechanical inactivation of ligninase is possibly the reason why ligninase accumulation is low or absent in agitated shake-flask cultures. Agitation seems to affect the catalytic activity of ligninase and has no apparent effect on either the rate of ligninase production or the physiology of P. chrysosporium. The detergents Tween 20, Tween 40, Tween 60, Tween 80, and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) are able to protect both purified ligninase and extant ligninase in culture fluids (free of biomass) against mechanical inactivation due to agitation. Addition of Tween 80 at the end of primary growth to agitated shake flasks containing either pelleted or immobilized mycelial cultures results in production and maintenance of high levels of ligninase activity over several days under conditions of high agitation. Possible mechanisms by which the detergents could protect ligninase are discussed. | |
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Effect of carbon dioxide and ethylene on berberine production and cell browning in Thalictrum minus cell cultures Y. Kobayashi, H. Fukui, M. Tabata | |
| Plant Cell Reports, Volume 9, Number 9 / Januar 1991, pp 496-499 | |
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Cultured cells of Thalictrum minus L. (Ranunculaceae), transferred from culture flasks to a bubble column bioreactor, produced little berberine and turned dark brown, even when supplied with sufficient oxygen. This phenomenon was ascribed to the removal of CO2 from the culture medium by bubbling air, and could be reproduced in flask cultures artificially deprived of CO2. The induction of cell browning by exogenously administered ethylene suggested that CO2 probably acts antagonistically against endogenously generated C2H4. The physiological damage caused by forced aeration could be prevented by adding 2 % CO2 to the air in the bioreactor. | |
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Effect of carbon source on cell growth and betalain production in cell suspension culture of Beta vulgaris M. Rodríguez Monroy, A. Jiménez Aparicio, G. Dévila Ortíz, G. Sepúlveda Jiménez | |
| Biotechnology Letters, Volume 16, Number 8, 1994, pp 853-858 | |
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In this work we studied the production of betalains by suspension culture of Beta vulgaris. The B. vulgaris grew at a doubling time of 4.8 days. The betalains production was found to be not-growth related. The culture's response to different carbon sources was investigated. Best growth and pigment production was obtained with sucrose. | |
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Effect of fatty acids on the mycelial growth and polysaccharide formation by Ganoderma lucidum in shake flask cultures. Yang F, Ke Y, Kuo S. | |
| Enzyme Microb Technol. 2000;27(3-5):295-301 | |
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Fatty acids were added into the media to investigate their effects on the mycelial growth and polysaccharide formation by Ganoderma lucidum. The experiments were carried out in freely suspended cultures or immobilized cultures using shake flasks. The results indicate that the extent of stimulation or inhibition were associated with the types and levels of fatty acids. Oleic acid at the level of 0.15 g/100 ml led to a significant increase in cell concentration from 0.20 to 0.46 g/100 ml in a suspended culture and palmitic acid was of great advantage to polysaccharide production. In contrast, linoleic acid (0.1 g/100 ml) drastically suppressed both mycelial growth and polysaccharide formation. In immobilized cultures with fatty acids, the stimulation of mycelial growth remained the same level, but the enhancement of polysaccharide production became less. In addition, the growth of G. lucidum in the pattern of immobilization might be beneficial to the production of mycelia and polysaccharide. | |
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Effect of oxygen limitation and medium composition on Escherichia coli fermentation in shake-flask cultures Losen, M., Froelich,B., Pohl, M., Buechs, J. | |
| Biotechnol. Progress. 20 (4), 1062-1068, 2004. | |
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Shake-flask cultures are widely used for screening of high producing strains. To select suitable strains for production scale, cultivation parameters should be applied that provide optimal growth conditions. A novel method of measuring respiratory activity in shake-flask cultures was employed to analyze Escherichia coli fermentation under laboratory conditions. Our results suggest that the length of fermentation, choice of medium, and aeration do not normally satisfy the requirements for unlimited growth in shake flasks. Using glycerol rather than glucose as a carbon source greatly reduced the accumulation of overflow and fermentative metabolites when oxygen supply was unlimited. A rich buffered medium, Terrific Broth (TB), yielded 5 times more biomass compared to LB medium but also caused oxygen limitation in standard shake-flask cultures at shaking frequencies below 400 rpm. These results were used to optimize the production of benzoylformate decarboxylase from Pseudomonas putida in E. coli SG13009, resulting in a 10-fold increase in volumetric enzyme production. This example demonstrates how variation of medium composition and oxygen supply can be evaluated by the measurement of the respiratory activity. This can help to efficiently optimize screening conditions for E. coli. | |
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Effect of oxygen supply on passaging, stabilising and screening of recombinant Hansenula polymorpha production strains in test tube cultures Stoeckmann C., Losen M., Dahlems U., Knocke C., Gellissen G., Buechs J. | |
| FEMS Yeast Research 4, 195-205, 2003 | |
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Twenty-four Hansenula polymorpha transformants were passaged and stabilised in glucose medium and screened in glycerol medium for recombinant phytase in shaken test tubes. The cultivations were performed under either limited or non-limited oxygen supply. Maximum oxygen transfer capacities of test tubes were assessed by sulfite oxidation. Oxygen-limited glucose cultures resulted in a partially anaerobic metabolism and formation of 4.1 g ethanol 1[-][1], which was subsequently aerobically metabolised. Non-limited oxygen supply led to overflow metabolism and to accumulation of 2.1 g acetic acid 1[-][1], reducing the biomass yield. The use of glycerol in the screening main cultures prevented by-product formation irrespective of oxygen supply. Preculturing in glucose medium under non-limited oxygen supply resulted in a 20-h lag phase of the screening main culture. This lag phase was not observed when preculturing was performed under oxygen limitation. Phytase activity was on average 25% higher in cultures passaged, stabilised and screened under limited oxygen supply than in cultures under non-limited oxygen supply. | |
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Effect of oxygen supply rates on growth of Escherichia coli
I. Studies in Unbaffled and Baffled Shake Flasks L. E. McDaniel, E. G. Bailey and A. Zimmerli | |
| Appl Environ Microbiol. 1965 January; 13(1): 109-114 | |
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The effect of oxygen-supply rates on bacterial growth was studied in commercially available unbaffled and baffled flasks with the use of Escherichia coli in a synthetic medium as a test system. The amount of growth obtained depended on the oxygen-supply rate. Based on oxygen-absorption rates (OAR) measured by the rate of sulfite oxidation, equal OAR values in different types of flasks did not give equal amounts of growth. However, growth was essentially equal at the equal sulfite-oxidation rates when these were determined in the presence of killed whole cultures. Specific growth rates were reduced only at oxygen-supply rates much lower than those at which the total amount of growth was reduced. For the physical set-up used in this work and with the biological system employed, Bellco 598 flasks and flasks fitted with Biotech stainless-steel baffles gave satisfactory results at workable broth volumes; unbaffled and Bellco 600 flasks did not. | |
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Effect of oxygen transfer rate on β-carotene production from synthetic medium by Blakeslea trispora in shake flask culture MANTZOURIDOU F. ; ROUKAS T.; ACHATZ B. | |
| Enzyme and microbial technology, 2005, vol. 37, no7, pp. 687-694 | |
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The effect of oxygen transfer rate (OTR) on β-carotene production by Blakelsea trispora in shake flask culture was investigated. The results indicated that the concentration of β-carotene (704.1 mg/l) was the highest in culture grown at maximum OTR of 20.5 mmol/(1 h). In this case, the percentage of zygospores was over 50.0% of the biomass dry weight. On the other hand, OTR level higher than 20.5 mmol/(1 h) was found to be detrimental to cell growth and pigment formation. To elucidate the effect of oxidative stress on β-carotene synthesis, the accumulation of hydrogen peroxide during fermentation under different OTRs was determined. A linear response of β-carotene synthesis to the level of H2O2 was observed, indicating that β-carotene synthesis is stimulated by H2O2. However, there was an optimal concentration of H2O2 (2400 μM) in enhancing β-carotene synthesis. At a higher concentration of H2O2, β-carotene decreased significantly due to its toxicity. | |
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Effect of Oxygen-Supply Rates on Growth of Escherichia coli
II. Comparison of Results in Shake Flasks and 50-Liter Fermentor L. E. McDaniel, E. G. Bailey, A. Zimmerli | |
| Appl Microbiol. 1965 January; 13(1): 115–119 | |
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Growth of Escherichia coli and chemical changes in the medium were very similar in highly baffled flasks and in a 50-liter fermentor run under the same oxygen-supply conditions, based on sulfite-oxidation rates. Flasks with stainless-steel baffles (Biotech) gave growth patterns and rates of glucose and NH4-N utilization almost identical to those of the fermentor; results with Bellco 598 flasks (with 6 to 7 mm deep indentations) were quite similar. Unbaffled and Bellco 600 flasks (3 to 4 mm indentations) were similar to the fermentor at very high and very low oxygen-transfer rates, but gave much less growth than the fermentor at intermediate levels. Maximal oxygen-uptake rates occurred in the fermentor at the end of the logarithmic-growth phase when growth was 40 to 75% of maximum. In the fermentor, both sulfite-oxidation rates and rates of oxygen uptake correlated reasonably well with the total amount of growth produced. | |
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Effect of scale-up on serpentine formation by Catharanthus roseus suspension cultures A. H. Scragg, P. Morris, E. J. Allan, P. Bond, M. W. Fowler | |
| Enzyme and Microbial Technology, Volume 9, Issue 10, October 1987, Pages 619-624 | |
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A culture of Catharanthus roseus has been developed that is capable of growth-linked serpentine formation. Two separate cell lines of this culture, C87 and C87N, were grown in air-lift bioreactors of 7, 30, and 80 liter working volume. Good growth was obtained with both cell lines in all vessels, with better growth rates at the higher volumes. In contrast, serpentine formation was very low when either cell line was grown in any of the vessels when compared with shake flasks. The reason for this loss of alkaloid formation does not appear to be associated with either bioreactor type or cell line. | |
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Effect of Shaking Speed and Type of Closure on Shake Flask Cultures L. E. McDaniel and E. G. Bailey | |
| Appl Microbiol. 1969 February; 17(2): 286–290. | |
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Growth of microorganisms and biosynthesis of microbial products in shake flasks may be limited by operating conditions which provide inadequate supplies of oxygen. Methods are described for meeting the oxygen requirements of test organisms by using standard baffled flasks with pad-type closures and shaking at relatively high speeds. Growth of Escherichia coli B in a rich medium and production of candidin by Streptomyces viridoflavus were the test systems used. Flasks shaken at 230 to 385 rev/min gave sulfite oxidation rates of 1 to 8 mmoles of oxygen per liter per min over a useful working volume range (40 to 150 ml in 300-ml flasks). These rates are as high as those obtained in agitated fermentors under usual operating conditions. | |
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Effect of small scale culture vessel type on hyphal fragment size and erythromycin production in Saccharopolyspora erythraea M.E. Bushell, G.L. Dunstan and G.C. Wilson | |
| Biotechnology Letters, Volume 19, Number 9 / September 1997 | |
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Erythromycin production dynamics in stirred, baffled shaken and non-baffled shaken flasks was strongly correlated with the different distributions of hyphal particle diameters observed. Production only took place when hyphal fragments with diameters greater than 88 mgrm were observed. Results are consistent with significant hyphal breakage rates, even in non-baffled shaken flasks. | |
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Effectiveness of orbital shaking for the aeration of suspended bacterial cultures in square-deepwell microtiter plates Wouter A. Duetz, Bernard Witholt | |
| Biochemical Engineering Journal, Volume 7, Issue 2, March 2001, Pages 113-115 | |
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Growth of heterogeneous culture collections in microtiter plates is advantageous for logistic reasons and also in enabling significant savings in medium costs, labor input and use of equipment during large screening projects. The main hurdles to overcome for aerobic microbial strains are the prevention of cross-contamination and excessive evaporation while assuring sufficient aeration rates. For this purpose we developed a sandwich spongy silicone/cotton wool cover to close the wells of square-deepwell microtiter plates. Oxygen transfer rates were derived from growth curves of Pseudomonas putida and were shown to be threefold higher during orbital shaking at a shaking diameter of 5 cm at 300 rpm (24 mmol O2 l−1 h−1 at a culture volume of 0.75 ml) in comparison to a shaking diameter of 2.5 cm. Photographic analysis showed a clear influence of the shaking diameter on the hydrodynamic behavior in the wells; during shaking at a 2.5 cm amplitude, out-of-phase conditions occurred resulting in poor vertical mixing, while a 5 cm shaking amplitude led to an optimal surface to volume ratio and a turbulent flow. | |
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Effects of aeration on tropane alkaloid production by transformed root of Atropa belladonna in flask cultures Kung-Ta Leea, Takashi Yamakawab, Tohru Kodamac, Yasuo Igarashia and Koichiro Shimomura | |
| Journal of Fermentation and Bioengineering, Volume 86, Issue 6, 1998, Pages 614-616 | |
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Transformed roots of Atropa belladonna strain M8 were cultured in shaking flasks aerated with normal air at 1 vvm, capped with aluminum foil, or capped with a silicon rubber stopper, to investigate the effects of aeration on root growth and on tropane alkaloid production and composition. After four weeks, the tropane alkaloid productivity of roots grown in flasks aerated at 1 vvm reached 52.9 mg/l, and the individual tropane alkaloid contents in the roots were, in mg/l dry wt., (−)-hyoscyamine 4.8, 6β-hydroxyhyoscyamine 1.4, littorine 1.2, and scopolamine 1.0. These results indicate that aeration at 1 vvm air has an important effect on the control of the tropane alkaloid composition in root cultures. In particular, the scopolamine ratio increased from 5.7 to 11.9% of the total alkaloids accumulated in the belladonna cultured roots. | |
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Effects of cultivating conditions on the mycelial growth of Ganoderma lucidum in submerged flask cultures F.-C. Yang, C.-B. Liau | |
| Bioprocess and Biosystems Engineering, Volume 19, Number 3 / September 1998 | |
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In this paper the effects of environmental conditions on the mycelial growth of Ganoderma lucidum were investigated in shake flask cultures. The optimal temperature and pH were found to be around 30-35v°C and 4, respectively, in a glucose-ammonium chloride medium. The maximum mycelial concentration reached to around 350 mg/100 ml. The formation of mycelial pellets and their ultra structure was demonstrated to be greatly affected by cultivating conditions. Increasing surface aeration would be beneficial for mycelial growth. However, too high rotating speed in shake cultures had a detrimental effect on the formation of mycelial pellets and the optimum was found to be 100 rpm. | |
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Effects of culture conditions on production of extracellular laccase by Rhizoctonia praticola Janusz G, Rogalski J, Barwi M, Szczodrak J | |
| Pol J Microbiol. 2006;55(4):309-19 | |
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It was found that the soil-dwelling fungus Rhizoctonia praticola 93A was capable to produce laccase in submerged cultures. Effects of culture conditions on the enzyme biosynthesis in shaken flask and aerated bioreactor cultures were evaluated to improve the yields of the process. Production of extracellular laccase was considerably intensified by the addition of Cu2+ to a carbon-limited and nitrogen-sufficient culture medium (C/N = 0.98). When an optimized medium containing glucose (2 g/l) and L-asparagine (1.5 g/l) was used and enzyme synthesis was stimulated by addition of 5 microM Cu2+ before inoculation, maximal laccase activities obtained in a batch cultivation were, approximately, 1000 nkat/l. Under these conditions, addition to the medium of the aromatic inducer 2,5-xylidine (1 mM) led to a 10-fold increase in laccase activity. Laccase productivity in shaken flask cultures was also enhanced (to more than 4000 nkat/l on day 3) by using a medium with the initial pH of 7.5. Such a high value of the optimal medium pH for laccase production by R. praticola is exceptional among the ligninolytic fungi. In fermenter fungal cultures supplemented with cupric ions, the highest laccase activity (about 4000 nkat/l after 3 days' cultivation) was reached after 24-h incubation using a bioreactor with the aeration rate of 21/min, the agitation speed of 200 rpm, and a constant medium pH of 8.0. | |
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Effects of Liquid Physical Properties on Oxygen Transfer in Penicillin Fermentation Fred H. Deindoerfer, Elmer L. Gaden, Jr | |
| Appl Microbiol. 1955 September; 3(5): 253–257 | |
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The success of fermentation processes often depends upon the maintenance of an environment sufficient in oxygen concentration so as not to limit or impair normal respiratory activity. Maintenance of such an environment is, in turn, largely dependent on the adequacy of oxygen transfer from air to the liquid medium. Of the possible pathways for oxygen transport, absorption into the medium and subsequent diffusion to the cells is undoubtedly the main one in submerged fermentation. Bartholomew et al. (1950b), Hixson and Gaden (1950), and Wise (1951) were the first to consider the mechanisms of oxygen transfer in fermentation systems, and they developed essentially identical methods for the quantitative expression of absorption rates. The absorption of oxygen into aqueous solutions, especially fermentation media, is generally considered as a liquid-film controlled mass-transfer operation. As such it should be expected that the mass-transfer rate, specifically the "oxygen absorption" rate for this case, is a function of those physical properties of the broth which affect interfacial area, turbulence, and boundary (film) conditions. Two such properties which undergo considerable change during many fermentations are surface tension and viscosity. For an excellent treatise on the subject of oxygen transfer in fermentations, the reader is referred to the recent review by Finn (1954). Apparently, no reports have been published of the changes in the rate of oxygen transfer which are due to physical property alterations of the broth as the fermentation progresses. Many fungal fermentations produce thick non-Newtonian broths as the mycelium concentrations rise, often as high as 25 to 30 g of dry tissue per liter during the fermentation. Metabolic activities, particularly hydrolysis of complex protein structures, utilization of carbohydrates, and the depositing of end-products in the broth, would be expected to change the surface tension considerably. The investigation reported here was undertaken in order to obtain information on the extent of changes in surface tension and viscosity during a penicillin fermentation and the degree to which they influence the rate of oxygen transfer from air to fermentation broth. | |
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Effects of oxygen and carbon dioxide on inosine fermentation Hiroshiro Shibai, Ayaaki Isizaki, Hiroshi Mizuno, Yosihio Hirose | |
| Agr. Biol Chem, 37 (1), 91-97, 1973 | |
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The effects of oxygen and carbon dioxide in inosine fermentation were investigated from the industrial viewpoint. Oxygen supply at the rate of more than 5x10-7 mol/ml min was indispensable for maintaining the high productivity of inosine in jar fermenters as well as in shaking flasks. Oxygen deficiency due to insufficient oxygen supply, on the other hand resulted in the inhibition of inosine production, even though glucose added to the medium was entirely assimilated. In addition to sufficient oxygen supply ventilation was also indispensable since an increased tension of carbon dioxide reduced the inosine production capability of the cells. | |
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Effects of pH on the water solubility and 1-octanol–water partition coefficient of 2,4,6-tribromophenol Kuramochi H, Kawamoto K, Sakai SI | |
| J. Environ. Monit., 2008, 10, 206 - 210 | |
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2,4,6-Tribromophenol (TBP) is expected to exist in both ionic and non-ionic forms in the environment due to ionisation of the phenolic group at near neutral pH. In this study, the water solubility (Sw) and 1-octanol–water partition coefficient (Kow) of aqueous solutions of TBP at various pH values were measured using the shake flask method. The ionisation resulted in increasing Sw and decreasing Kow by two to three orders of magnitude. From the experimental results, the environmental partitioning characteristics of TBP and the effect of pH on partitioning were discussed through a comparison with the properties of tetrabromobisphenol A (TBBP-A), which has two phenolic groups. Furthermore, the pH dependence of Sw and Kow was represented using a Henderson–Hasselbalch type model and the validity of the model was evaluated. The model was found to be highly useful for predicting the pH dependence within the range of pH 3 to 9. | |
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Effects of Position and Geometry of Current Pole on Mixing Characteristics in Shaking Vessel Kato, Hiraoka, Tada, Saito, Nomura | |
| JOURNAL OF CHEMICAL ENGINEERING OF JAPAN, Vol. 30 (1997) , No. 6 pp.1147-1148 | |
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In previous papers a center pole installed in a shaking vessel, called a current pole, is shown to be very effective for liquid mixing, particle dispersion and regeneration of hairy root. In this paper, the effects of position and geometry of the current pole on mixing characteristics are discussed. | |
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Empirical models for oxygen mass transfer: a comparison between shake flask and lab-scale fermentor and application to manganiferous ore bioleaching F. Veglio, F. Beolchini, S. Ubaldini | |
| Process Biochemistry, Volume 33, Issue 4, March 1998, Pages 367-376 | |
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An investigation on oxygen mass transfer in shake flasks and in a lab-scale bioreactor is reported. An analysis of the main factors influencing oxygen mass transfer in shake flasks has been carried out using full and fractional factorial designs. The factors investigated were: mixing conditions (rpm); temperature (°C); weight of closure (g); liquid hold-up (ml); and geometry of shake flasks. The ANOVA (analysis of variance) showed the importance of the investigated factors on the oxygen mass transfer coefficient. An empirical model with dimensionless parameters has been proposed in order to predict the oxygen mass transfer in several experimental conditions. An investigation has also been performed on the oxygen mass transfer in a lab-scale bioreactor, considering stirring, air flow rate and temperature as factors. Parameters of an usual empirical model for oxygen transfer coefficient have been estimated. The relationships proposed have been obtained in a wide range of experimental conditions, and they can be used both to determine if oxygen limiting conditions occur during biological tests in the shake-flask scale or in the lab-scale bioreactor, and in the phase of translation of experimental data to a bigger scale. Using this model the bioleaching of manganiferous minerals by heterotrophic microorganisms is judged to take place just in microaerobic conditions, i.e. in oxygen limiting conditions. | |
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Empirical models for oxygen mass transfer: a comparison between shake flask and lab-scale fermentor and application to manganiferous ore bioleaching Alvarez-Lorenzo C.; Castro E.; Gomez-Amoza J.L.; Martnez-Pacheco R.; Souto C.; Concheiro A.; Hallahan D.L.; West J.M.; Smiley D.W.M.; Pickett J.A.; Veglio F.; Beolchini F.; Ubaldini S. | |
| Process Biochemistry, Volume 33, Number 4, March 1998 , pp. 367-376(10) | |
|
An invertigation on oxygen mass transfer in shake flasks and in a lab scale bioreactor is reported. An analysis of the main factors influencing oxygen mass transfer in shake flasks has been carried out using full and fractional factorial designs. The factors investigated were: Mixing conditions (rpm), temperature (°C), weight of closure (g), liquid hold up (mL), and geometry of shake flasks. The ANOVA (analysis of variance) showed the importance of the investigated factors on the oxygen mass transfer coefficient. An empirical model with dimensionlesss parameters has been proposed in order ot predict the oxygen mass transfer in several experimental conditions. An investigation has also been performed on the oxygen mass transfer in a lab scale bioreactor, considering stirring, air flow and temperature as factors. | |
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Enhanced production of an extracellular protease from Beauveria bassiana by optimization of cultivation processes Yerra Koteswara Rao, Shih-Chin Lu, Bing-Lan Liu, Yew-Min Tzeng | |
| Biochemical Engineering Journal, Volume 28, Issue 1, 2006, Pages 57-66 | |
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Statistics-based experimental design, response surface methodology was employed to investigate the effect of medium components on the production of extracellular protease from Beauveria bassiana A1. The optimum medium composition for protease production was found to be (in %, w/v): 0.72 shrimp shell powder, 0.60 soy powder, 0.19 sucrose and 0.68 yeast extract. This medium was projected to produce, theoretically, 281.14 U/ml. By using this medium, an experimental maximum protease activity of 280.72 U/ml in 7 days cultivation verified the applied methodology. Further studies on bioreactor production of protease were investigated in a 5 l stirred-tank and 30 l airlift bioreactors. In 5 l stirred-tank bioreactor, a maximum protease activity of 238.77 U/ml was attained in 6 days of cultivation when aeration rate and agitation speed were controlled at 0.6 vvm and 150 rpm, respectively. On the other hand, in 30 l airlift bioreactor the maximum protease activity of 283.84 U/ml was found with the controlled aeration rate of 0.9 vvm operated in a bubble column mode. This activity in the 30 l airlift bioreactor was close to that obtained from the shaker-flask cultivation study (280.72 U/ml). However, the duration of cultivation period using 30 l airlift bioreactor was shorter than the corresponding duration obtained from the shaker-flask cultivation by 4 days. | |
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Enhanced production of elastase by Bacillus licheniformis ZJUEL31410: optimization of cultivation conditions using response surface methodology Qi-he Chen, Hui Ruan, Hai-feng Zhang, Hui Ni, Guo-qing He | |
| J Zhejiang Univ Sci B. 2007 November; 8(11): 845–852 | |
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Sequential methodology based on the application of three types of experimental designs was used to optimize the fermentation conditions for elastase production from mutant strain ZJUEL31410 of Bacillus licheniformis in shaking flask cultures. The optimal cultivation conditions stimulating the maximal elastase production consist of 220 r/min shaking speed, 25 h fermentation time, 5% (v/v) inoculums volume, 25 ml medium volume in 250 ml Erlenmeyer flask and 18 h seed age. Under the optimized conditions, the predicted maximal elastase activity was 495 U/ml. The application of response surface methodology resulted in a significant enhancement in elastase production. The effects of other factors such as elastin and the growth factor (corn steep flour) on elastase production and cell growth were also investigated in the current study. The elastin had no significant effect on enzyme-improved production. It is still not clear whether the elastin plays a role as a nitrogen source or not. Corn steep flour was verified to be the best and required factor for elastase production and cell growth by Bacillus licheniformis ZJUEL31410. | |
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Enlargement of the culture scale from flask to jar fermenter in the production of ribonuclease N1 by neurospora crassa: Effect of agitation intensity on enzyme production Takebe H, Takahashi J, Ueda K | |
| Journal of Fermentation Technology (1971), Vol. 49, No.12, pp 989-996 | |
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The effect of agitation intensity on the production of ribonuclease (RNase) N1 was investigated by incubating Neuropsora crassa 74A-Y152-M7 ylo under various cultural conditions. In a shaking culture, the net productivity of RNase N1 was much improved by putting a baffle plate in the cultural flask; the improvement was found to be closely related to the increase in the oxygen uptake rate of mycelia caused by the increase in the intensity of agitation. In cultivation using a 30L jar fermenter, the optimum agitator speed for the production of RNase N1 was 22 rpm, the enzyme production decreased in accordance with an increase or decrease in the agitator speed. The degree of dispersion of a water-insoluble liquid (a mixture of 4 parts of toluene with 1 part of carbon tetrachloride) in water was found to be a useful index for expressing the agitation intensity in relation to enzyme production; the same productivity (amount of enzyme produced/ unit weight of mycelia) of RNase N1 was obtained under cultural conditions of the same agitation intensity in the shaking cultures as well as in the aeration-agitation cultures using a 30L jar fermenter. For the enzyme production, it was not favorable to keep the agitation intensity unchanged throughout the cultivation. A milder agitation was found to be preferable for the production of RNase N1 in the logarithmic growth phase, and a more vigorous agitation was required in the stationary phase to obtain a better yield of RNase N1. The yield of RNase N1, which was 25 units/mL of culture fluid at the beginning of this investigation, wa | |