Continuous culture techniques

J. Monod, Continuous culture technique. Theory and applications, Ann. Inst. Pasteur Paris 1950, 79, 390-410. 

The yeast produced by continuous culture techniques is separated from the liquid medium and solvent washed by centrifugation or filtration techniques. After drying, a protein supplement is obtained, which contains 65-68% protein and is suitable for addition to animal feeds. This protein content compares very favorably with that of dry fish meal, which contains about 65%, and dry skim milk powder with about 32%. The SCP processes have operated on the thousands of tonne/year scale in the U.K., France, and Italy, but regulatory problems with facilities operating on unpurified gas oil feedstocks have caused some shutdowns [64]. Nevertheless, because of a cell mass doubling time of 2.5-3 hr and the efficient carbon conversion to protein of this technology, these developments deserve to be explored further. 

The difference between batch and continuous culture techniques with respect to the effect of product inhibition is that under the conditions of continuous cultures product is diluted, while in batch runs product is accumulated. Therefore, in batch cultures reaction rates eventually slow down. In chemostat cultures, however, oscillations of x and p appear due to periodic effect of, for example, pH control and/or permanent inflow and outflow of fresh medium. For the mathematical modeling and computer simulation of this problem, it is possible to formulate the following differential equations ... 

The continuous culture technique has been used successfully in bioconversions involving filamentous moulds [80]. This system may allow relatively higher ethanol production while enjoying some of the advantages of a continuous system. Different residence times for liquid and insoluble substrate in a continuous system are relatively easy to achieve, moreover cells attached to the substrate are also retained in this system [113]. 

The first culture technique, reported in 1927, to be attempted commercially was a surface-culture, shallow-pan technique, though this method has not been used for many years. Relatively soon after this, in 1933, production using a submerged culture technique was reported and this method has been in use continuously since then. Various significant developments have been made, notably the addition of caldum carbonate to neutralise the adds produced in order to increase yields (1937) and the use of sodium hydroxide for neutralisation (1952). 

Despite the advantages of continuous cultures, the technique has found little application in the fermentation industry. A multi-stage system is the most common continuous fermentation and has been used in the fermentation of glutamic add. The start-up of a multi-stage continuous system proceeds as follows. Initially, batch fermentation is commenced in each vessel. Fresh medium is introduced in the first vessel, and the outflow from this proceeds into the next vessel. The overall flow rate is then adjusted so that the substrate is completely consumed in the last vessel, and the intended product accumulated. The concentration of cells, products and substrate will then reach a steady state. The optimum number of vessels and rate of medium input can be calculated from simple batch experiments. 

Fig. 7. Scheme for a two-stage continuous cultivation technique. A = acid, 5 = base, M=medium, P = pump, S = substrate, Cl and C2 = base or acid flow, FI = culture flow, F2 = substrate flow, F3 = outflow, Z1 = medium flow... 

Continuous culture systems have been widely used to culture microorganisms for industrial and research purposes (Kubitschek 1970 Tempest 1970 Veldkamp 1976 Rhee 1980). In recent years, these culture techniques have found their way into the bioassay methods of ecotoxicology and allelopathy (Rhee 1980). The early development of a continuous culture system can be traced back to the work of Novik and Szilard (1950 a,b) who developed the first chemostat. In a continuous culture system, nutrients are supplied to the cell culture at a constant rate and to maintain a constant volume, an equal volume of cell culture is removed. This allows the cell population to reach a steady state, where the growth rate and the total number of cells/ml of culture remains constant. Two kind of continuous culture systems can be distinguished turbidostat and chemostat. ... 

Hybridoma Cell produced by the fusion of antibody-producing plasma cells with myeloma/carcinoma cells. The resultant hybrids have then the capacity to produce antibody (as determined by the properties of the plasma cells), and can be grown in continuous culture indefinitely owing to the immortality of the myeloma fusion partner. This technique enabled the first continuous supply of monoclonal antibodies to be produced. 

D. E. F. Harrison and B. Chance, Fluorimetric technique for monitoring changes in the level of reduced nicotinamide nucleotides in continuous cultures of microorganisms, Appl. Microbiol. 19, 446-450 (1970). 

The plant cell culture technique of micropropagation of flavour-producing plants will be able to help with their agricultural cultivation and will relieve the pressure on the wild populations. Micropropagation will be able to propagate those plants where conventional propagation is difficult or will be to multiply elite stock. This may be required if demand for natural flavours continues to increase. 

Flow cytometry [141, 142] is a technique that allows the measurement of multiple parameters on individual cells. Cells are introduced in a fluid stream to the measuring point in the apparatus. Here, the cell stream intersects a beam of light (usually from a laser). Light scattered from the beam and/or cell-associated fluorescence are collected for each cell that is analysed. Unlike the majority of spectroscopic or bulk biochemical methods it thus allows quantification of the heterogeneity of the cell sample being studied. This approach offers tremendous advantages for the study of cells in industrial processes, since it not only enables the visualisation of the distribution of a property within the population, but also can be used to determine the relationship between properties. As an example, flow cytometry has been used to determine the size, DNA content, and number of bud scars of individual cells in batch and continuous cultures of yeast [143,144]. This approach can thus provide information on the effect of the cell cycle on observed differences between cells that cannot be readily obtained by any other technique. 

By then Huzella had completely lost interest in my project which proposed that argyrophil fibers were artifacts made of this obscure polysaccharide called hyaluronic acid between the collagen fibers. Huzella never accepted this conclusion because he had absolutely no interest in polysaccharides. But, being a gentleman, he let me continue my work. It was important for my future work that I started in histology and learned tissue culture techniques, and through my research on hyaluronic acid, I also learned biochemistry. 

New cell culture techniques, which may improve the applicability of renal epithelial cultures, are also required. Currenfly there exist two commercially available cell culture perfusion systems, which allow the continuous perfusion of culture media and optimized oxygenation [243]. These systems allow stable longterm culture of quiescent adherent cells [244]. Continuous medium perfusion furthermore may lead to the re-expression of lost functions in continuous cell hues and the maintenance of differentiated properties in primary cells. Recently our laboratory has demonstrated that LLC-PKj cells maintained in a newly developed perfusion system (EpiFlow ) changed from a glycolytic to a more oxidative phenotype [72]. Evidence is also available from experiments in our laboratory that this mode of cultivation helps to prolong the lifetime of primary cultures of proximal tubular cells. Combining perfusion culture with co-culture of a cell type that is an anatomical neighbour in vivo (e.g. epithelial with endothelial, interstitial or immune cells) may improve the state of differentiation of both partner cells and increase the complexity of autocrine and paracrine interaction [73]. 

Research with anaerobic cultures can be in batch or continuous culture. Fed-batch anaerobic reactors are not known to us but may very well be feasible. A reliable technique for batch cultures uses serum bottles sealed with butyl rubber stoppers and crimp sealed with an aluminum cap. Anaerobic microorganisms in batch flasks are mainly cultured by Hungate s methods [7] that are widely accepted in the research community. We will not describe these techniques but will focus on the different continuous culture apparatuses that have been developed over the years. 

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