Abstract
Continuous culture techniques were developed in the early twentieth century to replace cumbersome studies of cell growth in batch cultures. In contrast to batch cultures, they constituted an open concept, as cells are forced to proliferate by adding new medium while cell suspension is constantly removed. During the 1940s and 1950s new devices have been designed—called “automatic syringe mechanism,” “turbidostat,” “chemostat,” “bactogen,” and “microbial auxanometer”—which allowed increasingly accurate quantitative measurements of bacterial growth. With these devices cell growth came under the external control of the experimenters and thus accessible for developing a mathematical theory of growth kinetics—developed mainly by Jacques Monod, Aron Novick and Leo Szilard in the early 1950s and still in use today. The paper explores the development of continuous culture devices and claims that these devices are simulators for standard cells following specific requirements, in particular involving mathematical constraints in the design and setting of the devices as well as experiments. These requirements have led to contemporary designs of continuous culture techniques realizing a specific event-based flow algorithm able to simulate directed evolution and produce artificial cells and microorganisms. This current development is seen as an alternative approach to today’s synthetic biology.
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Notes
Probably the first computer-based simulation in biology was carried out by Britton Chance, reported in a paper entitled The mechanism of catalase action. II. Electric analog computer studies in (1952).
It is also a story of transforming life into technology, comparable to the development Hannah Landecker revealed for tissue culture in medical research. “The life form of the cultured cell is a manifestly technological one: It is bound by the vessels of laboratory science, fed by the substances in the medium in which it is bathed, and manipulated internally and externally in countless ways” (2007, p. 3).
“The most important [requirement] is that the cultures should be constantly mixed, homogenized, and in equilibrium with the gas phase. This is achieved either by shaking or by bubbling air (or other gas mixtures) or both. Bubbling is often found inefficient unless very vigorous, when it may provoke foaming which should be avoided” (Monod 1949, p. 378).
Photomultipliers were used as counters in biology from the 1940s on, for instance for liquid scintillation, “in which the human eye was replaced by a highly sensitive, fast-responding photoelectric device for detecting and counting scintillations” (Rheinberger 2001, p. 148).
“It has been suggested that the difference between the two systems described above is that in the Chemostat or the Bactogen one is dealing with starved bacteria, while in the photocell controlled apparatus the bacteria are not starved” (Novick 1955, p. 101).
Novick and Szilard were interested in spontaneous mutation of a B strain of E. coli (B/1), “which is resistant to the bacterial virus T1, sensitive to the bacterial virus T5, and which requires tryptophane as a growth factor” (Novick and Szilard 1950b, p. 709).
“As the control system corrects the flow rate to maintain constant density, there will be variations in flow rate whose magnitude will depend on the sensitivity of the density-detecting system. The mean flow rate, however, will precisely equal the growth rate since the density N tends, in a properly adjusted machine of this type, neither to consistently increase or decrease” (Novick 1955, p. 99).
“The extent of application of continuous cultures in industry today is difficult to determine because this industrial information is not readily available in print […] Nevertheless, the available listing is impressive. Continuous cultures are used in the production of beer, sake, alcohol, and acetic and lactic acids; of vitamins (B12) and drugs (alkaloids); and of antibiotics (penicillin and chlortetracycline). Continuous cultures are also used for steroid conversion and the production of vaccines” (Kubitschek 1970, p. 9).
This delayed the introduction of continuous culture techniques to industry until the late 1950 s (Herbert et al. 1956).
An important improvement in the 1950s and 1960s for both domains, research as well as industry, was the maintenance of the artificial steady state for cells up to 4 months (Herbert et al. 1956). But the increase in duration came along with a serious problem, the selection of adhesive variants: so-called biofilms (wall growth). Biofilms are problematic because they falsify measurements of turbidity and prevent cells from dividing and evolving in long-term experiments. Thus, adventurous techniques were developed to avoid deposition in the culture chamber, among these a kind of “windshield wiper” (Anderson 1953, p. 734).
According to personal communication with Rupert Mutzel, the GM3 is currently the only device which succeeds in maintaining bacterial cells exclusively in suspension for such a long period.
The term “event-based algorithm” refers to a programming paradigm of information science in which the flow of the program is determined by events, e.g. inputs of sensors (Faison 2006). It is closely related to queuing theory, which has recently been used in synthetic biology “to investigate how ‘waiting lines’ can lead to correlations between protein ‘customers’ that are coupled solely through a downstream set of enzymatic ‘servers’” (Cookson et al. 2011, p. 1).
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I would like to thank Rupert Mutzel from the Freie Universität Berlin for supporting my study on continuous culture techniques.
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Gramelsberger, G. Continuous culture techniques as simulators for standard cells: Jacques Monod’s, Aron Novick’s and Leo Szilard’s quantitative approach to microbiology. HPLS 40, 23 (2018). https://doi.org/10.1007/s40656-017-0182-x
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DOI: https://doi.org/10.1007/s40656-017-0182-x