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Eduard Buchner (1860–1917).
Eduard Buchner (1860–1917).
Even at the end of he nineteenth century, there were still respectable scientists (including Louis Pasteur) who argued that there was something special about the chemistry of life, and that some ‘life force’ was involved in what were called vitalistic processes. The final refutation of this idea came in 1897, from the work of the German Eduard Buchner.
Buchner decided to tackle the question of fermentation, which divided opinion at the time. Fermentation is a process that takes place without using oxygen in which living cells convert foods such as sugar into simpler compounds, such as alcohol and carbon dioxide, and the energy that powers the cells is released. Fermentation is a common process in living things, but Buchner studied the simple process of alcohol production, which involves yeast. Fermentation was seen by many to be a physiological act inseparably linked with the life processes of yeast, although Friedrich Wöhler (see here) had poked fun at the vitalism idea, as far back as 1839, by satirically summarizing the argument as: ‘In a word, these infusoria gobble sugar, and discharge ethyl alcohol from the intestine and carbon dioxide from the urinary organs.’ Yeast is indeed a living organism, and yeast was essential for the process. But was this because the yeast cells were alive, or because they contained some chemical substance that encouraged (catalysed) the conversion of sugar into alcohol and carbon dioxide without any need for vitalism? Could fermentation occur without the presence of living yeast cells?
The only way to find out was by experiment. Buchner had long been interested in the problem, and when he was appointed as Professor Extraordinary for Analytical and Pharmaceutical Chemistry at the University of Tübingen in 1896 he was able to carry out the work on a large scale in a fully equipped laboratory. By 9 January 1897 he was ready to send his key scientific paper, Über alkoholische Gärung ohne Hefezellen (On Alcoholic Fermentation without Yeast Cells), to the editors of the journal Berichte der Deutschen Chemischen Gesellschaft.
Yeast cells are like small bubbles filled with a semi-liquid substance, the protoplasm, surrounded by a comparatively firm cell membrane. To investigate the chemical composition of the cell contents, the experimenters had to remove the membrane by crushing it, because if they used any chemically active solvents or high temperatures this would alter the chemistry they wanted to study. And it was important that the process should be completed as quickly as possible, to minimise the possibility of any change while the experiment was going on.
So Buchner started with living yeast cells, but then treated them to a series of processes which killed them and reduced them to their constituent chemical parts by purely physical means. These included mixing dry yeast cells, grains of quartz sand, and a soft crumbly rock (rather like pumice), known as diatomite, and then grinding the mixture, including the yeast cells, with a pestle and mortar. The mixture became damp as the yeast cells ruptured and their contents were released. Then, the damp mixture, which resembled a thick dough, could be squeezed to extract a ‘press juice’ used in the experiments.
This process was very efficient. Half a litre of liquid could be obtained from 1,000 grams of yeast, providing plenty of material to work with. When sugar solution was added to freshly pressed yeast juice, a strong production of gas occurred; in containers where the juice had been mixed with concentrated sugar syrup and left for several hours there was active frothing of carbon dioxide bubbles, and a thick layer of foam formed, showing that fermentation was occurring. When sugar was dissolved in juice at blood heat, these effects were visible within fifteen miniutes. Careful investigations by Buchner and his colleagues established that carbon dioxide and alcohol were produced in exactly the same proportions as in fermentation with live yeast. But microscopic investigation revealed no living yeast cells in the extract.
In further studies, Buchner found that the key chemical substance that promotes the breakdown of sugars in this way is an enzyme, which he called zymase. Zymase is a protein that is manufactured inside yeast cells, so in that sense life is involved in the process of fermentation. As Buchner put it in his Nobel Lecture (he was awarded the chemistry prize in 1907 for his biochemical researches and his discovery of cell-free fermentation), ‘the difference between enzymes and micro-organisms is clearly revealed when the latter are represented as the producers of the former, which we must conceive as complicated but inanimate chemical substances’. But the crucial point is that the chemistry carries on whether the yeast is alive or dead. Enzymes are crucial players in many biological processes, but it is now possible to synthesize enzymes chemically without biology being involved.
