DCY02 & DCY03: DCambic Isolates

DCY02 and 03 Fermentation in Progress

I am happy to report that isolating the yeasts from The Mad Fermentationist’s DCambic was a success. I have written about it previously and had some issues at first. A lot of stuff going on in life then kept me from the project for a few months but eventually I got the time to get it going again. I have been posting off and on about it on Twitter, but it’s time I document it here.

The key to success the second time was growing a liquid culture first from a sample Mike gave me, rather than plating it straight from the sample. After a few step-up culture iterations I was able to isolate four different yeasts from the beer, instead of just the one I got the first plating attempt. Two were useless for brewing: one is possibly Rhodotorula and the other is another yeast that does not ferment. The others were almost certainly Brettanomyces.

I can tell them apart on a plate because one (DCY02) grows white colonies and the other (03) tan colonies. Liquid cultures are also markedly different: DCY02 forms a pellicle very quickly, smells very funky and drops out fairly quickly, whereas DCY03 takes much longer to form a thinner pellicle, smells very fruity and drops out a lot slower. Taste profiles from the starter are along the same lines as the smell.

I made two simple extract beers in April (1.040 of pilsner DME) and the picture above is of fermentation about two weeks in. They were slow to start but that may be due to low pitching rates. In any case, I have not yet bottled these as they seemed to reactivate after I moved in late May. Perhaps just shaking them a bit roused enough yeast into suspension to do something, and I want to make sure they’re done before I bottle. (I will update this once they are done.)

Strain Descriptions

DCY02: The “white” isolate, based on colony appearance. This has a funkier Brett character with less of the fruity qualities. It produces a pellicle very quickly, and during fermentation produces a thick foam cap that looks more like Sherry flor than ale yeast kräusen. It dropped out of suspension faster than DCY03.

DCY03: The “tan” isolate. This is significantly less funky, and a lot fruitier. It doesn’t produce much foaming during fermentation, and in fact may appear to be doing nothing at all. If shaken, though, there will be a lot of CO2 released. It did not produce a pellicle until several months after pitching and left the wort cloudy for about a month.

Distribution

I hope to have more details soon, both from my beers but also from those of a few brewers I sent samples to. This was also the first time I’ve distributed anything so I kept it limited to four people. However, in the future I will release larger amounts to whomever wants it for a small fee to cover shipping and materials. Future announcements will be made on the blog and on Twitter.

PSA: Monitor your Yeast Bank

Much to my horror, recent attempts to revive four(!) different banked yeasts from my library have been unsuccessful. I fortunately have “backups” of the ones I consider most important, the “DCY” yeasts (which reminds me that I need to post an update about the DCambic yeasts, which are still fermenting one half-gallon each) and was able to work from that. But what I had considered my primary source of the two DCambic strains appears to be totally dead after just a few months.

I made a switch about six months ago to banking in isotonic NaCl solution after reading Samuel’s post on the matter. My previous attempts at reviving strains have succeeded, even with vials that were a few months older at the time. So, I’m suspecting operator error at this point and am very unwilling to cast this as a fault of the method. After all, Samuel used it for a while, and this is not a novel method. Possible problems include:

  • Improper solution: I used sea salt, because I couldn’t find a pure salt without additives. A lot contain minute amounts of dextrose, and I was trying to limit any sort of metabolism. Due to unknown levels of other salts in the mix it could require a different amount to be isotonic. I could also just have measured it wrong.
  • Storage temperatures at the back of the fridge were too low. Perhaps they dipped into freezing at times, and the small volume cooled off quickly.
  • I just moved a few weeks ago and somehow they got damaged in transit. Not likely, I don’t think, since I just moved within the same building and they were back in the fridge in just minutes.
  • Or, perhaps, I simply killed the sample while transferring into the salt solution. I had already revised my transfer method to avoid shocking yeast with a hot loop but it’s still a possibility.

The other two attempts were from slants. I have not had any issues in the past even with old slants, but these two for whatever reason opened slightly in storage and the tape around the cap wasn’t enough to hold the air in. They dried out as a result. I have successfully recovered yeast from such a slant before, but I am not so lucky this time–not yet, at least. As a last-ditch effort I will add sterile wort directly to the slant vial instead of picking off with a loop.

In either case, though, this highlights the need for monitoring things and perhaps something as simple as periodically tightening the caps on my vials. I may switch back to slants, at least temporarily, while I figure out what could be going wrong with the salt solution method.

Update: I was able to wake the two slanted strains up from their dead, dried, reddish state by adding sterile wort directly to the slant and shaking vigorously throughout the evening. In addition to having dried off, they were also slanted on media I made very early on that wasn’t ideal. I am surprised, frankly, that I was able to do this.

Update #2: Correcting update #1, it looks like only one of the slants actually yielded live yeast. As for the two DCambic yeasts from the NaCl storage medium, colonies finally are visible a week after plating. This is not unusual for Brettanomyces left at 20-22C/68-72F so this may have been much ado about nothing.

Baking with SD Isolate

Bread made with SD yeast isolate

I finally got around to doing something I’ve wanted to do since the beginning: bake bread leavened with the yeast isolated from a sourdough culture. A few days before baking I started growing a starter of sorts. I did three steps up from a very small amount of yeast stored in isotonic salt solution. This is similar to what I do for beer but I stopped at about 200mL of weak broth (can’t remember if it was YEPM or MYPG; I forgot to label the jar when I made it). Once I had enough and it had settled I used a sterile pipette to suck up the sediment from the bottom, about 6 grams’ worth of loose, liquid sediment for a 500g dough. It rose overnight in the upper 60’s with a proof of about four hours the next morning. The tiny amount of yeast is in line with what I’d use with dry baker’s yeast for a long rise–it’s probably equivalent to a sixteenth of a teaspoon or so of active dry yeast. The dough is just flour, water, salt and yeast.

I figured it’d not be worthy of a post because it’d probably turn out exactly like a bread made with standard baker’s yeast. After all, it’s just yeast, with no lactic acid bacteria to create the sourness. To a great extent that is true, but the neat thing is that this yeast apparently produces a ton of diacetyl. I had noticed this before when using the sourdough culture it came from (my wheat starter) but I had for some reason assumed it was bacteria doing it. But when I took it out of the proofing basket today I got a massive whiff of butter. This made for a quite nice aroma while baking, although it seems to have completely disappeared in aroma and taste by the time the bread cooled.

These yeasts were the fastest growing (at least in terms of visible colonies) that I’ve seen so far. I suppose that’s consistent with what you’d expect from a continuously-refreshed culture like sourdough, but that’s just speculation. I have no idea what species they are, but I’m fairly certain it’s not S. cerevisiae based on the quick growth and colony characteristics. The colonies spread out much more than I’ve seen with S. cerevisiae. They are also whiter and flatter. I keep meaning to ferment wort with it to see its alcohol tolerance and sugar utilization but never get around to it.

Sourdough Plating #3 and DCambic Update

This is the third and final attempt at plating out the whole sourdough starter. I did a few things differently:

  • I fed the three sourdough starters and let them ferment for 12 hours before starting. Last time two of the starters were right out of the fridge.
  • I put even less of each culture in the sterile water to dilute it
  • I used bromocresol green + potato dextrose plates, plus one potato lactose plate

The rye starter’s plate grew mold in a few days. It totally took over before I had a chance to see anything else growing. The other two starter’s plates plus the lactose plate (on which I plated the wheat starter) were totally fine. I let them grow out for 16 days.

Clockwise from top left: brown rice flour starter, wheat flour starter, wheat starter on potato lactose plate

The colonies look translucent and shiny from certain lighting angles, but they are off-white, smooth and round and actually matte. I did not see any flat colonies like last time. The weirdest surprise is the almost identical growth on the lactose plate. I am going to assume that this is due to whatever sugar was in the potato and not the lactose. My goal in doing so was to see if I could get the bacteria to grow on there and was not expecting the yeast to grow at all. You can see very tiny colonies on each of the plates between the large yeast colonies and smaller yeast colonies. They are transparent and shiny. I believe these are the lactic bacteria.

I think I may not have used enough bromocresol green, or the plates were old enough that it had already started to fade. I was expecting green colonies here, too, but they were white. On my first (recent) attempt I did see green colonies but the plates were also darker back then. The colonies looked quite similar between the two starters. This is a change from the first time I did this (earlier in the year) but could be because I am using a different medium.

I picked out a yeast colony from each and plated it on YEPM medium (recipe from jaapie’s site; the ingredients are not cheap or easy to find for hobbyists like me). In the past, the yeast I isolated from the wheat starter didn’t appear to ferment maltose whereas the rice starter’s did. We’ll see if these grow on it (from what I can tell, yeasts generally but not always can grow on something they can ferment and vice-versa).

On another note, I finally resumed the DCambic project from April. I had isolated a yeast from it back then but it appeared to not do much besides make lots of strong fruit esters. Notably, it didn’t seem to produce any alcohol. This time I instead went for growing it in liquid first instead of plating directly from the sample. I now seem to have two different types of yeast in liquid, judging from the two colors of sediment I see. A darker sediment formed first. I then started shaking to aerate the culture on a regular basis for a few days and then added this to more sterile wort, which I similarly shake on a regular basis. I am now seeing a lighter color sediment forming as well. I also see lots of evidence of fermentation, though I did not see any krausen at any point. I shook up the culture yesterday and streaked a sample on a PDA plate. I’m hopeful that this time I can get the yeast that produces the distinct Bretty aroma I can smell from the culture.

Sourdough Plating Results

These are the results after 5 days of growth.

Wheat flour starter (Carl’s Friends) on top. Bottom left is the brown rice flour starter (homegrown) and the right is the immature rye flour starter.

I am a bit surprised for a few reasons. First, from looking at them I was convinced that there was no yeast growth. At first I had all three of them in a big plastic bag, and the smell was not at all yeasty. It also looked like all of the colonies on the wheat and brown rice starters were very glossy like bacterial colonies. I had previously plated both of those on malt extract agar and the yeast colonies looked very different from each other, but definitely looked like yeast. I saw nothing of the sort here. The rye starter definitely had nothing that looked like yeast, but it is still young.

The first surprise came when I opened the tops to photograph them. I first took some of the colonies from the wheat starter plate and put them in a bit of light wort. But when I took a whiff both the wheat starter plate and rice starter plate smelled very yeasty. The smell from the bag I mentioned before was from the rye starter plate. It smelled not wholly unpleasant. I could pick out acetic acid but there were other things underneath. The colonies were tiny and slightly yellow. I’m not sure if they were heterofermentative LAB that produce acetic acid or some sort of enteric bacteria.

In the end I tossed out all the plates. But now looking at the picture above I can see that the colonies aren’t equally glossy. On malt extract agar (a long time ago) the wheat starter’s yeast were flat, dull and bigger than brewer’s yeast. That kind of matches some of the colonies I see in the picture. The rice starter’s yeast looked and behaved a lot like brewer’s yeast (I planned on trying it for brewing but never got around to it. I just made some small liquid cultures.) I don’t see anything of the sort here.

Later today I’m going for a third try on the bromocresol green and dextrose plates. Hopefully I can get some distinct yeast growth since LAB aren’t supposed to grow on it.

Sourdough Project False Starts

The sourdough stuff I promised in the last post hasn’t gone too well so far. I made the plates I mentioned but doing things with them has not gone well.

The idea is to do a few things:

  • Compare the organisms in three sourdough starters
  • Evaluate the properties of the yeasts I can isolate
  • Try to learn a thing or two about lactic bacteria
  • Try to cultivate a new starter (the third from above) from scratch, which I hadn’t done in a while

So the fourth bullet is sort of working, but not without some problems. I made an effort to use sanitized glasses and silverware to handle the starter so that I could be reasonably sure the organisms were coming from the flour and not from my environment. It took a long time but I can’t say whether that is due to that or some other reason. After a lot of strange stages (producing what I think is DMS, since it smelled a lot like cooked corn) it looks like it is becoming a viable starter. However, I messed up one day and stuck an unsanitized fork in there. It was just starting to turn around, but it sped up its turnaround after that. Having recently worked with other starters it could be I “contaminated” it with known-good organisms.

(As a side note, I always wonder if the number of environmental bugs is sufficient to overcome whatever is in the flour, but I suspect that is highly complicated and not subject to a simple yes/no assessment. I wish I had access to irradiated flour so I could put it to some sort of test.)

The first three require me to plate the starters. My first effort at this was destined to fail: I tried to streak after dipping a needle in the starters. I got some bugs from the early 2nd day stage of my new starter, some bacteria I can’t identify (yellow colonies on PDA). The other plates grew only a handful of yeast colonies but the plates grew tons of mold before I could do anything else with them. In any case, the bits of liquid dough along the streaks made it quite difficult to tell if things were growing at first.

I repeated the effort this unplanned long weekend, since I was home from the storm. I went with spreading a sample of sterile water that had a bit of starter dipped in it. After 24 hours there is no growth at 75F. We’ll see what happens in the next couple of days.

Not dead yet!

Despite the total lack of posting, I am not anywhere near done with the blog. As I mentioned in the last post I had a three week trip followed by moving, and it took until the end of August to settle down. Plus, I also got engaged.

I am preparing some potato plates today or tomorrow. I have an idea for some posts about sourdough. So, stay tuned!

Pellicle

As you may have guessed from my posting frequency, I’ve not done much with yeasts recently. A flurry of unfortunate events in the past month has kept me busy, and with an upcoming international trip followed by a move when I get back there will be little going on until August or so. So, I will leave you with the only interesting thing happening lately, which is that the sample I inoculated with a few drops of the DCambic grew a pellicle.

It’s actually grown two pellicles now. The first fell when I moved the bottle, disappearing completely in a few hours. This one grew a week or so later and also fell, but not before covering the entire surface. The neat thing is that you can see the sunken pellicles at the bottom of the flask.

In addition to the pellicle, I’ve also noted a few things. First, there is a layer of dark sediment which I also noticed in the vial Mike gave me. Some sort of yeast, perhaps, but I have no way of knowing right now. Second, about a week or so before the first pellicle showed up I started seeing CO2 bubbles, which I had not seen before. The yeast ring around the flask came from that stage. All of this almost two months after the initial inoculation. I will have to repeat this after I move and then try to isolate the bugs from there instead of from the vial like I did the first time.

DCambic Update #2

It looks like nothing else is going to grow on the plates, so I went ahead and streaked out one of the colonies on another plate and inoculated a small liquid sample. I will step that up as usual. The plate had a slightly funky smell unlike any yeast plates I’ve dealt with so far. The colonies also look bigger, glossier and more irregular than others I have dealt with. I have not yet worked with laboratory Bretts, though, so I can’t compare. In any case, given that the previous liquid sample grown from the raw beer developed an intense Bretty character I am quite sure that is what I’m dealing with here. I’m not too good at describing the various Brett flavors, so I’ll venture only that it is fruity and phenolic. Although there was no visible fermentation there is definitely alcohol in the aroma. I am imagining the new liquid sample from the pure culture will smell the same.

I had originally planned to streak out two plates, one with potato dextrose and one with potato lactose, but I ended up not having the time to prepare them this weekend. I went instead with another malt extract plate. This doesn’t help me get any new information on how the strain behaves but it’ll do for now. When I have the time I will prepare those potato plates with bromocresol green and restreak. One nice thing about potato media is that you can easily swap the sugars and make different types from the same batch of potato infusion.

Nothing at all grew on the brilliant green plate. My previous experiments with them had at least some yeast growth but it was very slow. This time I don’t see anything. It is safe to say my experiment with that medium is a failure. Good thing it was unnecessary for this experiment.

DCambic Plate Growth

It’s now been about a week since I plated the DCambic sample. A few days ago I began to see growth of what appear to be yeast colonies growing on the malt extract plate. They are round with small peaks in the middle, and appear to be a little glossy. It’s hard to tell right now because I am not opening the plates just yet to avoid contamination. I want to leave these out for at least three weeks to allow any slow-growing organisms to appear.

There is also some yeast growth in the liquid sample. I began to see a layer of sediment a few days ago. No pellicle or visible signs of fermentation, but the pH dropped to the mid 4 range (using strips to check) and there was a somewhat Bretty aroma. There is a little funk but the dominant smell is sort of fruity, not unlike canned peaches. I am stepping that up to 250mL as we speak.

Once I’m done with incubating I will streak out the individual organisms and grow them in liquid. It’ll be interesting to see if I get the same characteristics as in the current liquid sample.