Last semester I began to incorporate bits of the scientific history of Microbiology into the content expectations for this course. Many students of the sciences become very cognizant of current concepts and techniques in their chosen field, but this many times comes at a cost. I find that understanding of many complex concepts is therefore facilitated and eased with the appreciation for how those concepts were developed in the field. Consequently, I introduced during the past year the “Famous Microbiologist” segment, where we might learn about some of the giants of the field who are unfortunately given short shrift in our current textbook.
Today’s Microbiologist is Nobel Laureate Eduard Buchner (1860-1917.) Prof. Buchner changed the way that we think of the abilities of cellular life, and with a very clever series of experiments ushered in the new scientific discipline of biochemistry. Prior to Buchner’s experiments, the concept of vitalism maintained that biological activities required a living cell to occur. For instance, it was well known that the sugar from grapes could be converted into alcohol by yeasts, via the process of fermentation. Grape juice in the absence of yeast was unable to be converted into alcohol, and it was thought fermentation required some vital aspect of a living cell to occur.
A number of enzymes had been described during the 19th century which were able to carry out their activities outside of a cell. One very important enzyme that had early industrial use was chymosin, or rennin. This enzyme was initially extracted from the dry stomachs of calves, and was used in the cheesemaking industry to cause the coagulation of milk in the initial formation of curds. This reaction was relatively simple, transforming a single reactant to a product in one step. The process of fermentation converts glucose to ethyl alcohol via a number of steps. The number and the complexity of the chemical reactions was believed to be unable to occur outside of the cell.
Buchner began experimenting with ways to use cell-free extracts in order to reproduce the process of fermentation outside of the cell. Two technical issues had to be circumvented before he could be successful with this. First, he needed a way to effectively break open the yeast cell to release the cytoplasmic contents, and this was not a trivial issue. Fungal cells are surrounded by a thick cell wall that protects the cell from physical stresses. Second, he needed to release the cellular contents in such a way that they were protected from degradation and remain active once they were released. Since fermentation requires the activity of 9 independent enzyme activities in order to produce the final product, a breakdown at any one of the steps would result in no product being formed.
Buchner was able to overcome this technical hurdle by starting with a preparation of dry yeasts. Preparation of active dry yeasts was a direct result of innovations in the beer and wine brewing industries in the mid-1800’s, and offered to Buchner a starting material that was easy to work with in the laboratory, but still maintained cellular enzymes in a non-degraded state. He then took the dried yeasts, added finely ground glass fragments, and ground them with a mortal and pestle. He noticed at this point that the dried material became moist, as the cellular contents of the yeasts were released. The broken yeast cells were then put into a powerful press, and squeezed through a mesh to remove the cellular debris, and the liquid portion was collected. When the liquid portion was examined under the microscope, no cells were observed, and when this material was put onto solid media plates, no colonies of yeast grew. Buchner took this liquid fraction, added solutions of simple sugars to them, and found that carbon dioxide and ethanol could be observed to be produced as the mixture sat for several days, in the absence of any living cells.
Buchner’s conclusion from this simple experiment was that yeast cells secreted enzymes that accomplish this process into the environment, however the enzymes that carry out fermentation actually are not secreted and are found in the cytoplasm. When he broke the cells by grinding with glass particles, the cellular contents were released. His work was novel, as it was the first description that a complex biological process (the conversion of simple sugars like glucose into ethanol) could be accomplished by cell free enzymes, and initiated a trend to reduce these processes to discrete steps. His work was recognized by his being awarded the Nobel Prize in Chemistry in 1907.
Bonus Time! Based on what you know from Bio I and the material so far in this course, suggest a reason that Buchner’s experiments might have failed miserably. That is, if he had done something just a bit differently in the lab, why might he have not observed fermentation?