Capturing wild yeast to be able to use them for brewing (also known as bioprospecting) had been a project that I have had in my mind for a few years. I had been reading about it and I knew it was something that demanded patience and that it was not easy, with a high chance of having multiple failures before having a good result. But it was also something that could be fun and, well, I could always try to refresh my memories from microbiology classes back at the University years ago. The thing is that, among the workshops announced for the Spanish Homebrewers Association of 2019 in Bilbao, there was one with the following title: “Hunting wild yeasts”. It was like a signal to start taking this project seriously. The workshop was delivered by Aitor Lekuona, a microbiologist specialized in fermentations and brewing science, with an impeccable presentation that I loved. His advice, along additional information that I gathered from the internet (basically from the essential Milk the Funk wiki) were the pillars in which I based my “hunt”. In addition, I was able to have a chat with Aitor after the workshop and, after telling him about my intentions to capture some wild yeasts for brewing, he offered to help me to solve any doubt I could have. Of course, I did not miss the opportunity and, when I did not know what to do, I contacted him and he kindly advised me. Because of this, first of all I would like to thank him because if it was not for his help, maybe this project would have failed a few months ago.
Fruits selected for this first try at bioprospecting
Almost of the sources agree that spring and summer are the best times of the year to capture wild yeasts. As usual, I postponed everything for a few months until an afternoon in mid-September, when the summer of 2019 was in its last days, I spent some time collecting fruits from which I hoped I could get some interesting microorganisms. The possibilities are endless (fruits, flowers, bark,…) but I decided to pick some apples and some blackberries. In the case of apples, just because I had read that they were a suitable fruit for yeasts and, in the case of blackberries, it was just a matter of availability. For any sample you pick, you have to be careful and try to pick fruits that have not been treated with chemicals as pesticides and the like. In my case, after a walk in the forests around my neighborhood, I collected some samples from red apples that grow wild without any human intervention, a few blackberries and some green apples from trees that grow also without any chemical treatment. I collected all of these samples in 50 mL sterile centrifuge tubes.
Once I collected the fruit samples, to try to grow the yeasts in them, I decided to do it with three different methods: the one described by Aitor Lekuona in the workshop I previously mentioned, one developed by David Thorton from Southyeast Labs and the one proposed in the Sui Generis Brewing blog. Next, I will give a little description for each one of them.
Growth in the three samples following the method described by Aitor Lekuona
In the method described by Aitor Lekuona you start with a wort with an original gravity around 1.020. This wort must be boiled to sanitize it, without adding hops. Once the wort is cooled down, 20-25 mL of it are poured in 50 mL sterile tubes containing the samples. You let it ferment for a minimum of 2-3 weeks, shaking and opening the caps a little bit to let carbon dioxide from fermentation escape. After a few weeks, you can streak agar plates to purify strains.
The method developed by David Thorton starts with a wort of similar original gravity, 1.024, that must be also boiled without hops. In this case, adding yeast nutrients is recommended. 75 mL of this wort are transferred to a 250 mL flask and then the sample is added, in this case the fruit. Then put it on a stirplate, covering the top of the flask with some aluminum foil. Let it ferment for 48 hours. As soon as froth appears, put an airlock on the flask while keeping it in the stirplate. After another 48 hours, decant and discard the liquid and keep the biomass. Add 75 mL of fresh wort, this time of an original gravity between 1.032-1.040 and again with some nutrients, and purge with carbon dioxide. Keep the flask in the stirplate with the airlock until fermentation is finished. Then decant and discard the liquid again and keep the biomass. In the last step, purge again with carbon dioxide and add another 75 mL of wort, this time with an even higher original gravity, 1.083. Do not forget to add some yeast nutrients too. Put the airlock on and keep the flask in a stirplate until the gravity drops around 1.008. Finally, you can purify strains in agar plates if you want. This method is designed to capture mostly Saccharomyces yeasts and lactic acid bacteria.
The third method is the one recommended in the Sui Generis Brewing blog. Start with 250 mL of a wort with an original gravity between 1.045-1.065, hopped to around 10-25 IBUs and with 0.5 mL of phosphoric acid to reduce pH. Boil to sanitize, cool and add about 35 mL of cheap vodka. The low pH should inhibit the growth of pathogenic bacteria, hops should inhibit lactic acid bacteria and alcohol should avoid the growth of a lot of microorganisms that do not tolerate alcohol. Let it ferment with an airlock to avoid mold and acetic acid bacteria.
Flask after the second step of the Thorton’s method
That is enough for theory. In practice, I started with around 250 mL of a wort of 1.024 original gravity from malt extract that I could use for both the Thornton and Lekuona methods. For the Sui Generis Brewing method, I made a wort of 1.050 original gravity, again from malt extract. To this wort, I added 0.5 mL of 10% phosphoric acid. I boiled it for about 10 minutes with some Northern Brewer hops to got about 25 IBUs. Finally, after cooling it down, I added 35 mL of vodka.
I transferred 20-25 mL of the first wort (1.024 O.G. without hops) to each of the three sterile tubes which contained pieces of red apple, 3-4 blackberries and pieces of green apple, respectively. I kept these tubes well closed. In addition, I added 75 mL of the same wort to a 250 mL flask, previously sterilized. I added some red apple pieces to this wort, just because it was the sample of which I have the greater amount (and also because I have a feeling that it could be my best bet). I covered the top of the flask with some aluminum foil and let it ferment. Finally, I transferred the hopped wort with vodka to a previously sterilized 500 mL flask. This flask received also some red apple pieces and then I put an airlock on it. It was time to wait for the magic to happen.
The next day, intrigued by expectation, I went directly to see how everything was. There was no visible activity in both flask. However, in the 50 mL tubes something was fermenting. After shaking them and loosening the caps a little bit, some hiss confirmed that some carbon dioxide had formed.
Two days later, from the 50 mL tubes the carbon dioxide continued to escape every time I loosened the caps. Besides, the three of them had some froth. I considered that they were already “inoculated”, so I removed fruits from them as aseptically as possible, avoiding this way the growth of molds. In the flasks, nothing interesting yet after three days.
Pellicle in the flask of the Sui Generis Brewing method
After a week and a half, in the bottom of the tubes whatever which had grown started to sediment. In that moment I noted what I thought were phenolic aromas in the tubes and, in the case of the tube inoculated with the green apple, some interesting esters too. As for the other two methods (I had put the airlock in the flask of the Thorton method a few days ago) I was supposed to remove the liquid from the flask of the Thorton’s method. Although it was clear that it was some fermentation activity, visually there was very little amount of biomass, so I opted for adding 75 mL of a fresh wort of 1.040 original gravity with some nutrients without removing the liquid. However, I did remove the fruit pieces from the flask when I added this fresh wort. In the flask of the Sui Generis Brewing method, when I was ready to dump it, some signs of activity showed. This activity increased and the next day a fine pellicle with quite big bubbles had formed.
The next days, the amount of biomass in the bottom of the 50 mL tubes was quite big. Or at least that was what I thought. For the Thorton method I had to perform the third step. This time I removed the liquid because after stopping the stirplate, a nice amount of biomass could be seen at the bottom of the flask (see picture above). I purged with carbon dioxide and added 75 mL of fresh wort with an original gravity of 1.083 plus some nutrients. I put the airlock on the flask and kept it on the stirplate. A couple hours later a krausen formed in the surface.
Several weeks passed without me being able to do anything apart from taking a look once in a while. Even though the pellicle that had formed in the flask of the Sui generis brewing blog got me amped, I think I should had tried something with it earlier (inoculate some wort, purify strains in agar plates,…), but I had no time to do it. Furthermore, I was not convinced with the aroma coming out the flask, and the fruit was still in the flask. So I decided to dump it, ending this part of the experiment. However, I think the method is valid and it could give good results and I have no doubts that I will use it again in the future. I still had two parts of this wild yeasts hunt experiment going on.
Everything set to streak agar plates
A couple of months after I collected the fruits I took some time to streak some plates with the samples from the tubes to purify strains. At this point, I asked Aitor Lekuona for advice, since I did not know if I should take the sample for streaking with or without shaking the tubes. He told me that probably the population of yeasts was big enough without shaking, but he recommended me to take two samples of each tube, one without shaking them and one after shaking them and letting them stand for 30 seconds. This way, the variety of microorganisms could be greater. So that is what I did.
I had prepared the agar plates a few days before following the instructions from (again) the Sui Generis Brewing blog (its “Your Home Yeast Lab” section is awesome!!). I had kept them several days in the fridge, where nothing had grown, so maybe I had made them correctly. I streaked two plates for each sample (before and after shaking the tubes) from the three tubes “inoculated” with red apple, blackberries and green apple, respectively. I also streaked two plates for the sample of the Thorton method, again before and after shaking the liquid. I left the plates at room temperature to see the growth.
Around the same time, thanks to an event organized by our homebrew club, Legamia, I was able to inoculate what had grown inside the flask of the Thorton method, without purifying, since I did not dump the liquid after streaking plates. The event consisted in brewing a batch of wort in a commercial brewery (thanks to Tito Blas brewing for this), with a recipe that the members of the club had designed (75% pale ale malt and 25% wheat malt, 1.050 O.G. and 25 IBUs). Then the wort was divided and each one of the club members would ferment it as they liked. I had 20 liters of wort and I took 5 liters for this experiment. I inoculated these 5 liters of wort with a mini-starter that I made from what had grown in the flask of the Thorton method (the biomass that was left from a starter made with 250 mL of wort with 1.035 original gravity). It took it a few days to start, at least to show some signs of fermentation, but finally a nice white krausen formed. Fermentation seemed to go well and, at bottling, final gravity was 1.005 (5.9% ABV) and pH was 3.8. It did not look bad at all. I tasted it at bottling and it was not bad, some cidery flavor, a little bit tart, but quite pleasant. However, as my homebrew club colleagues could attest, after carbonating it was barely drinkable. A lot of solvent off-flavors, a lot of acetic acid… A complete disaster. I wonder if exposing the beer to oxygen at bottling had something to do with this. Or maybe it was just the evolution of the beer and the microorganisms in it. Anyway, a little failure.
Growth in the agar plates
However, I had still the agar plates, so I could keep on trying. As you can see in above picture, all the plates showed some growth and each of them had some kind of purified strain. In one of them, probably because my technique was not aseptic enough, some mold growth was observed. I kept this plates in the fridge for a few months because I was not able to find some time to play around with them. When finally I found some time, I had some serious doubts that, starting from a single colony, I could get something to grow and obtain something good, but I tried anyway. To do this, I went back to Aitor Lekuona’s presentation. Then I selected which, with the naked eye, I thought were yeast colonies, considering their morphology. Medium size, a more or less rounded shape, white or pinkish color and quite opaque.
Before picking this colonies, I prepared five sterile tubes to which I added about 25 mL of sterile wort with an original gravity of 1.040, prepared from malt extract. I kept these tubes with the wort in a pressure cooker with the cap loose for 15 minutes at maximum pressure. After cooling them down, it was time to go for unique strains. As aseptically as I could, I selected those colonies which I thought seemed the best for my purposes. I inoculate each of the five 50 mL tubes with 25 mL of sterile wort with a different strain. Of these five unique strains, a couple of them came from the sample of green apples (a white one and a pinkish one), one white colony from the sample of blackberries, one pink colony from the sample of red apple and another white colony also from the sample of red apples but with the Thorton method.
Growht in tubes from unique strains
Fortunately, after the first day signs of fermentation were present, with carbon dioxide escaping the tubes when loosening the caps a little bit. This activity increased with time and in the third day I had to be careful when loosening the caps so the liquid and foam would not spill outside the tubes. After five days growing, I decided to go a step further and see the behavior of these strains in a more or less real environment, but on a small scale. My idea was to prepare 1 liter of hopped wort with an original gravity around 1.050 and with 20-25 IBUs and divide it in four equal parts to brew four mini-batches. It was four because I discarded one of the tubes because I found the aroma a little bit off. The four final candidates were the two colonies from the green apples, the colony from the blackberries and the colony from the red apples obtained with the Thorton method.
After inoculating each one of the four 500 mL bottles (previously sanitized) with a different strain, after 9 hours the four airlocks were bubbling. After 18 hours, all of them showed a nice krausen, specially the one of the strain from the red apple obtained with the Thorton method, that reached the neck of the bottle. I forgot to take a picture of that moment, but in picture below you can see the remainders of what was the krausen in each bottle. I left the bottles a few days more at room temperature, around 20ºC (68ºF) to let them ferment completely. In all, 11 days passed, when I took some samples to measure final gravity and pH. And of course, to taste the results of these mini-batches.
Mini-batches of four differnt unique strains
Next, you can see a summary of my notes from this tasting session for the four samples:
SAMPLE A (green apple – white colony)
pH 4.68; DF 1.014; 4.79% ABV / Good aroma, apple-like. It has also some hints of juniper/pine. Nice flavor. Good flocculation.
SAMPLE B (green apple – pinkish colony)
pH 4.79; DF 1.015; 4.63% ABV / Little aroma of any type. It has some herbal notes. Quite clear, good flocculation.
SAMPLE C (blackberries – white colony)
pH 4.73; DF 1.016; 4.46% ABV / Low aroma. Good flocculation, clearer than samples A and B. The flavor is not nice, somewhat strange.
SAMPLE D (red apple – white colony obtained with Thorton’s method)
pH 4.62; DF 1.012; 4.95% ABV / Similar in appearance to the other three samples. More aroma than samples B and C and little less than sample A, but nicer, fruitier. Nice flavor, quite clean.
Once I finished tasting and measuring all of them, I discarded samples B and C (pinkish colony from green apples and white colony from blackberries) for not having pleasant enough organoleptic properties. In addition, these two samples were also the ones with the less attenuation and higher pH values of the four. In fact, pH values seemed to me a little bit high in all cases comparing with usual values for a brewing Saccharomyces strain. Anyway, I kept tubes from samples A and D (white colony from green apples and white colony from red apples) so I could continue to test them. Both of them seemed promising to me, especially the one from red apples. My idea is to brew a small batch, around 3 liters, for each one of them, then pitch a starter, let it ferment, bottle and carbonate, to see if they can offer something that could be interesting for future brews. It would be very exciting to have a nice beer fermented with wild yeast captured by me.
This is all for now in my project of bioprospecting. I hope you find it interesting and, if some of you are doing something similar, please feel free to share your thoughts. I will keep on telling you how this project follows and if these strains are good enough to keep them. It has been a long entry, but I thought that it would be better to put everything in a single post. It has been a lot of months since I started until now and although it takes a lot of work, I must say that I am pretty happy with how everything went so far. It was very entertaining, I learned a lot of new things and, although I had some small disappointments, so far the project outcome is very positive. I thought about doing something similar in 2020 and, in fact, I collected some samples (flowers and cherries this time), but seeing that I had a limited free time I decided to discard these samples and focus in the samples that I have already. I will try to capture some new wild yeast in 2021.