In
the seventeenth century, a Dutch tradesman named Antoni van Leeuwenhoek
developed high-quality lenses and was able to observe yeast for the first time.
In his spare time Leeuwenhoek used his lenses to observe and record detailed
drawings of everything he could, including very tiny objects, like protozoa,bacteria, and yeast.
Leeuwenhoek discovered that yeast consist of globules floating in a fluid, but
he thought they were merely the starchy particles of the grain from which the
wort (liquid obtained from the brewing of whiskey and beer) was made (Huxley
1894). Later, in 1755, yeast were defined in the Dictionary of
the English Language by Samuel
Johnson as "the ferment put into drink to make it work; and into bread to
lighten and swell it." At the time, nobody believed that yeast were alive;
they were seen as just organic chemical agents required for fermentation.
In the
eighteenth and nineteenth centuries, chemists worked hard to decipher the
nature of alcoholic fermentation through analytical chemistry and chemical
nomenclature. In 1789, the French chemist Antoine Lavoisier was working on
basic theoretical questions about the transformations of substances. In his
quest, he decided to use sugars for his experiments, and he gained new
knowledge about their structures and chemical reactions. Using quantitative
studies, he learned that sugars are composed of a mixture of hydrogen, charcoal
(carbon), and oxygen.
Lavoisier was also interested
in analyzing the mechanism by which sugarcane is transformed into alcohol and
carbon dioxide during fermentation. He estimated the proportions of sugars and
water at the beginning of the chemical reaction and compared them with the
alcohol and carbon dioxide proportions obtained at the end. For the alcoholic
reaction to proceed, he also added yeast paste (or "ferment," as it
was called). He concluded that sugars were broken down through two chemical
pathways: Two-thirds of the sugars were reduced to form alcohol, and the other
third were oxidized to form carbon dioxide (the source of the bubbles observedduring
fermentation). Lavoisier predicted (according to his famous
conservation-of-mass principle) that if it was possible to combine alcohol and
carbon dioxide in the right proportions, the resulting product would be sugar.
The experiment provided a clear insight into the basic chemical reactions
needed to produce alcohol. However, there was one problem: Where did the yeast
fit into the reaction? The chemists hypothesized that the yeast initiated
alcoholic fermentation but did not take part in the reaction. They assumed that
the yeast remained unchanged throughout the chemical reactions.
Yeast Are Microorganisms
In 1815 the
French chemist Joseph-Louis Gay-Lussac made some interesting observations about
yeast. Gay-Lussac was experimenting with a method developed by Nicolas Appert,
a confectioner and cooker, for preventing perishable food from rotting.
Gay-Lussac was interested in using the method to maintain grape juice wort in
an unfermented state for an indefinite time. The method consisted of boiling
the wort in a vessel, and then tightly closing the vessel containing the
boiling fluid to avoid exposure to air. With this method, the grape juice
remained unfermented for long periods as long as the vessel was kept closed.
However, if yeast (ferment) was introduced into the wort after the liquid
cooled, the wort would begin to ferment. There was now no doubt that yeast were
indispensable for alcoholic fermentation. But what role did they play in the
process?
When more
powerful microscopes were developed, the nature of yeast came to be better
understood. In 1835, Charles Cagniard de la Tour, a French inventor, observed
that during alcoholic fermentation yeast multiply by gemmation (budding). His
observation confirmed that yeast are one-celled organisms and suggested that
they were closely related to the fermentation process. Around the same time,
Theodor Schwann, Friedrich Kützing, and Christian Erxleben independently
concluded that "the globular, or oval, corpuscles which float so thickly
in the yeast [ferment] as to make it muddy" were living organisms (Barnett
1998). The recognition that yeast are living entities and not merely organic
residues changed the prevailing idea that fermentation was only a chemical
process. This discovery paved the way to understand the role of yeast in
fermentation.
Pasteur Demonstrates the Role of Yeast in
Fermentation
Our
modern understanding of the fermentation process comes from the work of the
French chemist Louis Pasteur.
Pasteur was the first to demonstrate
experimentally that fermented
beverages result from the action of living yeast transforming glucose into
ethanol. Moreover, Pasteur demonstrated that only microorganisms are capable of
converting sugars into alcohol from grape juice, and that the process occurs in
the absence of oxygen. He concluded that fermentation is a vital process, and
he defined it as respiration without air (Barnett 2000;
Pasteur 1876).
Pasteur
performed careful experiments and demonstrated that the end products of
alcoholic fermentation are more numerous and complex than those initially
reported by Lavoisier. Along with alcohol and carbon dioxide, there were also
significant amounts of glycerin, succinic acid, and amylic alcohol (some of
these molecules were optical isomers — a characteristic of many important
molecules required for life). These observations suggested that fermentation
was an organic process. To confirm his hypothesis, Pasteur reproduced
fermentation under experimental conditions, and his results showed that
fermentation and yeast multiplication occur in parallel. He realized that
fermentation is a consequence of the yeast multiplication, and the yeast have
to be alive for alcohol to be produced. Pasteur published his seminal results
in a preliminary paper in 1857 and in a final version in 1860, which was titled
"Mémoire sur la fermentation alcoolique" (Pasteur 1857).
In 1856, a man named Bigo sought Pasteur's help
because he was having problems at his distillery, which produced alcohol from
sugar beetroot fermentation. The contents of his fermentation containers were
embittered, and instead of alcohol he was obtaining a substance similar to sour
milk. Pasteur analyzed the chemical contents of the sour substance and found
that it contained a substantial amount of lactic acid instead of alcohol. When
he compared the sediments from different containers under the microscope, he
noticed that large amounts of yeast were visible in samples from the containers
in which alcoholic fermentation had occurred. In contrast, in the polluted
containers, the ones containing lactic acid, he observed "much smaller
cells than the yeast." Pasteur's finding showed that there are two types
of fermentation: alcoholic and lactic acid. Alcoholic fermentation occurs by
the action of yeast; lactic acid fermentation, by the action of bacteria.