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A recent conversation about Rafael Arroyo has led to a guest post by Cory Widmayer! If you do not know Cory, most of the great fermentation ideas presented on this blog over the past numerous years have been his. Cory is an extremely capable microbiologist and fermentation chemist adept at isolating organisms and optimizing ferments. Our conversation was about how Arroyo’s experience with ABE fermentation fed his ideas about rum production. Arroyo still pops up in 21rst century scholarly work regarding Clostridia.
Cory is an idea factory, and despite the budget of mere hobbyists, we have been able to bring many of them to life. Fission yeast use is regaining traction in the spirits industry and we have logged hundreds of inquiries over the years. GI’s have been rewritten to allow for these yeast! Most of our present conversations regard importing ideas from industrial fermentations to develop aroma driven spirits as well as designing our own pilot plant equipment. Some of the ideas I am most excited about regard small logistical tweaks to encourage broader consortiums of ambient yeasts to unlock bound aroma in fruit fermentations; we are more than just rum! Cory and I believe the potential of most substrates is squandered! My bet is Cory will be a leader in turning much of that around.
Rafael Arroyo and the Golden Age of Industrial Fermentation
When I offered my help to Stephen over seven years ago, I was convinced that much of what we were reading in Studies on Rum could be brought to life through our raw determination and shoestring budgets. I still believe this, though I have been rightly humbled by the demands of our project. Things have been moving slowly but steadily, though I’ve decided to remain more in the shadows until I have compelling things to share.
The hardships I faced with building a lab and conducting experiments in my apartment eventually led to a fulfilling change in career—I’m now a fermentation scientist at LanzaTech, studying gas-fermenting Clostridia. As fate would have it, Rafael Arroyo was something of a Clostridium specialist, and I coincidently learned more of his career and the conditions that led to his Studies on Rum through my own study of gas fermentation. Stephen encouraged me to summarize some of what I learned for the blog, so here it is:
The science of fermentation gained unprecedented importance during the First World War when it was discovered that certain Clostridia could produce acetone, a chemical in high demand for the manufacture of explosives. No cheap petrochemical route existed at the time, making fermentation the most economical option. Chaim Weizmann, who would later go on to become the founder of Israel, developed the “ABE” process, or “acetone-butanol-ethanol” fermentation using select Clostridia.
Industrial fermentation with anaerobic bacteria was a novel idea for the time. Unlike fermentation with yeast, most Clostridia are strictly anaerobic and will perish in the presence of oxygen. ABE fermenters also require a relatively high starting pH (5.5-7) that demands sterility or at least pasteurization to control contamination. In addition to these challenges, ABE fermentation is a complex process producing a mixture of products that can be dramatically altered by fermentation conditions. Since interest and investment was strong, scale up of production became high priority throughout the world. Brilliant minds were drawn to the cause, bringing rapid development and cutting-edge techniques to the process. Consequently, this would open doors to many other advanced industrial fermentation regimes, ranging from acid production (such as propionic and butyric) to antibiotics and enzymes. The Golden Age of industrial fermentation had begun.
Figure 1 : Metabolic illustration of the biphasic batch ABE fermentation process – Initially, an acidogenic phase occurs resulting in acetic and butyric acid until the pH drops and substrate inhibition occurs. Then, sporulation is triggered, and the bacteria shifts into a solventogenic phase, driving conversion of the remaining sugar, acetate, and butyrate into ethanol, butanol, and acetone.
In the 1930s, Puerto Rico was a struggling agricultural island with little industrial development or opportunity. Through FDR’s New Deal, a generous stimulus was brought to the island that included an ABE fermentation plant. The ABE plant was built to work in conjunction with a local sugar mill, using molasses as feedstock. The Puerto Rico Research Station led by Rafael Arroyo was enlisted to prospect for ABE organisms and establish a fermentation process there.
Arroyo isolated a remarkable organism, Bacillus tetryl (later renamed Clostridium tetryl), from the roots of sugarcane. Arroyo claimed it was a facultative anaerobic Bacillus, though it may have been an aerotolerant Clostridium (no other facultative anaerobic Bacillus with the ABE pathway has been discovered). Aerotolerance is a rare quality for Clostridia, which could allow for a less intensive propagation by avoiding anaerobic conditions. While it couldn’t invert sucrose, it was found to grow and ferment well in inverted molasses. It also produced an unusually high ratio of butanol, which had become a priority over acetone after the First World War. Arroyo’s first claim to fame was patenting this organism and his fermentation process.
The demanding research and technical prowess required for ABE made Arroyo’s team in Puerto Rico a formidable one. Generous government funding and the incentive for development in Puerto Rico gave Arroyo the opportunity to expand and apply his expertise to new fermentation endeavors, such as the industrial production of acids (butyric and propionic), alcohol, wine, brandy, and rum.
Arroyo’s strong intellectual background led to creative new approaches to rum making, and his past with ABE was undoubtedly fundamental in the development of his philosophy. His emphasis on prospecting for and evaluating microorganisms was particularly valuable, as well as his skill in executing controlled experiments. Access to a top-notch analytical laboratory allowed for careful interpretation of fermentation results and sensory appraisal of distillates. Not to mention, cutting-edge equipment such as the super-centrifuge allowed for more efficiency and opportunity in process design. Progress was rapid and many groundbreaking publications were made, with Arroyo’s suave tone giving the impression that his conclusions were obvious, and his approaches were simple and affordable for the average distillery (we now know that this couldn’t be further from the truth!).
It is fascinating to comprehend the remarkable accomplishments Arroyo and his team made. To start, they were aware of Percival Greg’s experiments with S. pombe and adapted a more refined version with strict pH control as their primary fermentation strategy. They isolated their own strains of fission yeasts and tested them in elaborate fermentation schemes, discovering which strains were winners and how to induce them to produce coveted volatiles such as rum oil (C-13 norisoprenoids) and esters. The isolation of C. saccharobutyricum (possibly a Bacillus) and the development of a synergistic co-fermentation with fission yeast to make Jamaican-like heavy rum is a remarkable feat even by today’s standards. Isolation of an “Oidium” (Dipodascaceae) from tree slime flux and adapting it for use in Grand Arôme rhum was similarly extraordinary (this has been successfully emulated at Line 44 Distillery in New Zealand!). Moreover, they found the most efficient ways to conduct fermentation of molasses for rum, alcohol, and bacterial products, publishing several relevant patents.
Figure 2: A recent study with select Bacillus strains for fermentation of molasses to produce butyric acid, hydrogen gas, and biomass. There can be high variability in metabolism even between strains of Bacillus, as not all strains of B. thuringiensis and B. subtilis seem to be facultative anaerobes or capable of producing butyrate. These strains show a similar metabolite profile to what Arroyo described for his C. saccharobutyricum.
Government stimulus for R&D did eventually run out for the experimental station, and as the 1960s were approaching the Golden Age of Fermentation was coming to an end. ABE fermentation would almost completely disappear from the world (though there is a small resurgence happening now). Arroyo died young, and much of his work had drifted into obscurity.
Few distilleries in the modern world have enjoyed the privileges that Arroyo’s team had earned. Truly, we can say that no distillery in existence today is directed by a fermentation prodigy, with the support of a remarkably skilled team, high-tech equipment, and a generous budget. Marketing and practicality continue to lead the way in the spirits world, which is understandable—the surge in micro-distilleries throughout the world provides ample evidence that beverage distilling can be lucrative with a simple approach to fermentation.
So, why do I continue to study unusual yeasts in my spare time? Part of it comes from the joy of being a scientist and bringing fascinating ideas to life. Another part is that I admire quality and can visualize how spectacular spirits can be with a more meticulous approach to fermentation. Working with Stephen has been a joy, and I see no reason for it to end—I think we’ll get it someday.
Figure 3 : A recent fission yeast isolate from cacao beans. I was playing around with media to induce sporulation, and
had great success in this case.