Arroyo: Mitogenetic Radiation as a Factor in Industrial Fermentation, 1945

Follow along: IG @birectifier

A scientist across the world was asking me for this missing paper by Rafael Arroyo. The topic was covered in Studies on Rum and I was not aware a separate article was published in the journal Sugar in 1945. Nearly a decade ago, I wrote a brief sloppy post summarizing what you found at the time when you googled the topic.

All these years later, what is surprising is that there are still no distilleries in the world capable of duplicating Arroyo’s work and no one currently uses a simultaneous culture of fission yeast & saccharobutyricum. Giants like Bacardi could probably not duplicate the work. Lallemand has the skills, but not the will. The closest anyone has gotten to Arroyo’s simultaneous cultures is Line 44 in New Zealand, employing both fission yeast & aroma producing mildew yeast (and the results are sensational!).

Cory & I wondered over the years if mitogenetic radiation was ever a part of traditional rum fermentations, but there are no indications. I was also told there are some theories of why some labs historically could not duplicate the findings or found intermittency and that may have been from various types of background noise like radio waves or electromagnetic interference interrupting the communication. It would probably not like wifi…

There is a prize to be won by anyone that can duplicate this work. You’d probably get an article in Nature followed by headlines relaying around the science world. If all you found was no radiation, but a beautiful rum, you’d have the beautiful rum. This is likely to stay a scientific curiosity for years to come.

---

 

Mitogenetic Radiation As a Factor in Industrial Fermentation

Experiments in Puerto Rico with yeast cultures support controverted theory of Gurwitsch’s rays
By Rafael Arroyo, Ch.E.,S.E.

By “mitogenetic radiation” is meant an energy in the form of rays which is given up by some biological materials in certain stages of development. These rays pass through quartz, not through glass, and when they encounter other growing tissues in certain stages of development they may be recognized in the form of increased growth or reproductive activity. Alexander Gurwitsch published (1) his discovery of these so-called mitogenetic rays in 1923. Since that time, many articles dealing with the subject have appeared, some confirming and other failing to corroborate Gurwitsch’s claims. We were led to delve into the phenomenon of mitogenetic radiation as an incident to researches on rum manufacture started nine years ago (1935). In our work we followed a modification of the technique first described by Baron (2), who worked in Gurwitsch’s laboratory, and our results confirm Gurwitsch’s work.

(1) Gurwitsch, A. Die Natur des Spzifischen Erregers der Tellteilung. Arch. Mikrosk. Anat. und Entro. Mech., 100: 11:40 (1923).
(2) Baron. M.A. Uber Mitogenetische Strahlung bei Protisten. Roux. Arch. Entwichlungs Mech. Organ., 108: 617-623 (1926).

In the course of the work in rum manufacture we used mixed cultures of yeast and bacteria under strict biological control. The individual cultures consisted of pure strains of Schizosaccharomyces pombe (Lindner) and Clostridium Saccharo-Butyricum, the latter discovered and isolated by the writer. When fermentation was accomplished by the pure yeast acting singly in the substrate, it was observed: (1) fermentation and cell multiplication were very slow, especially during the first 24 hours; (2) most of the cell multiplication and practically all of the alcohol formation took place during the second half of the fermentation period, which usually lasted for 96 hours; (3) yeast cell counts conducted immediately after inoculation, and at optimum multiplication, proved that the original concentration of yeast cells usually increased from 15 to 20 times.

Whenever mixed pure cultures of yeast and bacteria were used under controlled conditions, we invariably obtained altogether different fermentation results. (1) The duration of the fermentation period was reduced to a very great extent; i.e., from 96 hours to a period of from 30 to 36 hours. (2) the yeast developed consistently a very high increase in activity, both as to power of multiplication and zymogenic action. Most of the alcohol was then produced during the first 24 hour period. (3) Yeast cell counts conducted immediately after seeding and at optimum development showed that the increase in cell concentration was no longer in the ratio of 1:15 or 1:20, but that ratios of even 1:50 were obtainable; and sometimes even this high value was surpassed.

This unexpected and curious phenomenon aroused interest in its cause. A careful study of the metabolic products of the bacteria when working in pure culture in a substrate similar to that used for the rum experiments was undertaken. It was found that, apart from the gaseous products, carbon dioxide and hydrogen, the main product of fermentation was a mixture of normal butyric acid, acetic acid, and propionic acid. Of the total mixed acids thus found, the normal butyric amounted to 95%, the acetic to 4.1% and the propionic to 0.9%. None of these products, particularly the one produced in greatest quantity can be regarded as an enhancer of alcoholic fermentation or an activator of yeast growth and reproduction. Nevertheless we studied the effect upon the yeast of the metabolic products of the bacteria minus the bacteria themselves.

To accomplish this, two sets of experiments were devised. In the first set we added to a series of yeast mashes a different individual amount of fermented bacterial mash in which the bacteria themselves had been killed by heat treatment. In this way the yeast cultures could receive the effects of the products of the bacteria, but the influence of the living organisms themselves was omitted. The results of these experiments indicated no favorable effects on yeast cell activation, on multiplication, or on the rate of alcoholic fermentation. Adverse effects were encountered as the percentage of added acids was increased, until in those cases where the total organic acid concentration amounted to 0.5% or more of the yeast mash weight, fermentation was greatly retarded or refused to take place.

In the second set of experiments, both the effects of the living bacteria and the organic acids produced by them were eliminated. To the yeast mashes were added different amounts of the slops obtained after driving away by distillation all of the acids produced in the fermented mash. By so doing we were allowing any other products of the bacterial metabolism of a non-volatile nature, freedom to exert their beneficial influence (if any) upon the yeast’s growth and multiplication, as well as upon its zymogenic power. As in the previous experiments, the yeast cultures failed to show any of the phenomena previously encountered during the mixed cultures fermentation experiments.

These series of experiments demonstrated that the activation of the yeast observed during our mixed yeast-bacteria fermentation work depended  primarily upon a certain influence exerted by the living bacteria and not upon any of their products of metabolism. This conclusion led to the suspicion that mitogenetic radiation might be the cause of the observed phenomena. Experiments therefore were conducted to prove whether this was the case. In these experiments, pure quartz tubes were used to irradiate the yeast culture through their walls, thus eliminating actual contact and symbiosis of yeast and bacteria. When the yeast mash was thus irradiated by the bacterial culture through the quartz wall, the phenomena previously observed again became manifest. In these tests no other effect than that of irradiation through the quartz wall was possible, so the positive results obtained became added proof that mitogenetic radiation was the activation agent responsible for the behavior of the yeast culture in the rum fermentation studies with mixed cultures, of which one was the culture of Clostridium saccharobutyricum. Further confirmation was obtained when an ordinary glass test tube was substituted for the quartz, for then the bacterial culture failed to activate the yeast through the ordinary glass wall.

Two more experiments, at intervals of eight days, were then performed, using the mixed culture fermentation technique previously employed in the rum studies. A molasses mash of 15.46° corrected Brix, 5.75 pH value, and total sugars content of 9.88 grams per 100 ml., was prepared and divided among six one-liter Erlenmayer flask, each containing 400 ml. of mash. After thorough sterilization, the flasks were inoculated with 10% seed of a pure culture of yeast. One of the flasks was used as a control and the other five were numbered in series, 1 to 5. Initial cell concentrations were determined. Flask No. 1 was immediately submitted to bacterial radiation from a pure culture of Clostridium saccharobutyricum. Nos. 2 to 5 inclusive were also submitted to the bacterial radiation in succession at intervals of seven hours. A total fermentation period of 30 hours was allowed, counting from the time of yeast inoculation. When this period was over, small samples were taken from each flask for pH determination, and the fermentation was immediately stopped in all flasks by the addition of a predetermined amount of 20% sulphuric acid solution. Determinations were then made on the fermented mash in each flask of (1) final Brix; (2) final yeast cell concentration per milliliter of mash; (3) total alcohol produced; (4) residual sugars, in terms of grams per 100 ml. of mash. The results are given in the tables.

From these experiment it may be concluded: (1) the performance of the cultures submitted to mitogenetic radiation was superior in all cases to that of the check; (2) both cell multiplication and alcohol formation increased as the period of exposure to the effects of the rays increased, but only up to a limit lying between 14 and 21 hours of irradiation; (3) the cultures exposed to the influence of the mitogenetic rays shows higher final cell concentrations per milliliter of mash, more alcohol formation, more weight of dry yeast per unit volume of mash, less residual sugars, and greater attenuation than the controls. These results seem to indicate that the phenomenon encountered during our rum researches when working with mixed yeast-bacteria cultures was due to the effect of mitogenetic irradiation from bacteria to yeast. They appear to confirm Gurwitsch’s theory of mitogenetic radiation, a much discussed scientific subject that has remained unsettled.