Sunday, February 12, 2012

Jacques Monod and the study of bacterial growth

Among the great scientists of the 20th century, Jacques Monod holds a prominent place. Together with François Jacob and André Lwoff he contributed to the creation of molecular biology through his study of genetic regulation, in particular the lac operon in E. coli. Before this achievement, which was awarded a Nobel prize in 1965, Monod helped define the nature of bacterial growth in a variety of experiments that took place during his doctorate. He published it in 1942, exactly seventy years ago, under the title "Recherches sur la croissance des cultures bactériennes" (a second edition, the one I read, was published in 1958).

In his experiments, Monod followed the growth of liquid bacterial cultures in flasks at controlled conditions of temperature and oxygenation. The turbidity of the culture, which was used as a surrogate value for bacterial biomass, was measured with a nephelometer (in laboratories, today, a spectrophotometer is usually employed, but the principle is the same). Bacteria were grown in complex (brain broth) or defined media with variable sources of carbohydrates. Monod used Bacillus subtilis and Escherichia coli (that he calls B. coliBacterium coli I suppose, which is the ancient taxonomy). 

Most notably, Monod showed that bacterial growth is limited by the amount of nutrients, but that the yield is independent of the concentration of these nutrients. The growth rate, however, is dependent of the nutrients' concentration and rapidly reaches a limit. What is more, this maximal growth rate can vary a lot with different carbon sources and temperatures. Today, we still talk about 'Monod kinetics' when we describe the growth of bacterial cultures.

Here's a typical growth curve (below), which we still represent the same way today. Above, we see how the growth rate changes in function of time. The exponential growth phase, during which the growth rate is constant (III), is characteristic of bacterial growth. What it means is that the bacterial population doubles at a constant rate. With E. coli, it can be as fast as a doubling every 20 minutes.

J. Monod, Recherches sur la croissance des cultures bactériennes. Hermann, Paris, 1958.

Seventy years later, not much has changed in the way we look at bacterial batch cultures!

In the second part of the book, Monod describes the phenomenon of diauxic growth (he coined this term, which is still in use today). Diauxic growth takes place when a mixture of different carbohydrates is used. In certain cases, bacterial growth shows two distinct phases separated by a stagnation period. Monod rightly explains that this phenomenon is due to the inhibition of certain enzymes. 
What we see in the figure below is two normal growth curves (a, b) and four diauxic growth curves (c, d, e, f). When you mix together glucose and rhamnose, for instance, the enzymes necessary to attack rhamnose are not active until all glucose is used, hence the biphasic curve. The mechanism of this inhibition, Monod tells us in his book, is unknown. It will be explained later as catabolite repression, a type of genetic regulation that prevents the production of non necessary enzymes, and with the lactose operon as standard example. 
J. Monod, Recherches sur la croissance des cultures bactériennes. Hermann, Paris, 1958.
Monod's work has long become standard knowledge in microbiogy. The beauty of these simple experiments remains as vivid as seventy years ago. In his book, Monod humbly wrote: "Peut-être le résultat le plus notable de ce travail sera-t-il de permettre d'utiliser avec plus de sûreté et de précision les courbes de croissance pour l'étude de la physiologie bactérienne". I would translate it as: "Maybe the most notable result of this work will be to allow us to use the growth curves with more certainty and precision for the study of bacterial physiology". Certainly it did, and still does.

References: Jacques Monod (1958). Recherches sur la croissance des cultures bactériennes.  (Deuxième édition.) Éditions Hermann, Paris.

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