Isolating Yeast from a Honduran Vineyard

I'm currently taking some of the last basket-weaving classes required to finish up my undergraduate degree in Microbiology. One of those classes offered a short trip to an agricultural school in Honduras to assist with harvesting their vineyard and subsequent wine making. Having just read through the Bootleg Biology website; I was inspired to make an entirely different project out of it.

I set out to isolate a wild yeast strain from the grape skins in the vineyard we were working in. In case you weren't aware, the white powdery film on the fruit in grocery stores isn't some pesticide or preservative - it's yeast. They float around in the air outside until they eventually encounter something sweet. If that something happens to be a fruit, then they wait there for the skin to eventually break open so they can nom on the sugary juice. You might ask, "Why not just isolate a wild yeast from a local vineyard? Or one of those fruit farms that lets you go pick blueberries in the summer?" Rest assured, I will. But at the time it happened to be December, and I was going to a vineyard in Honduras anyway.

So I brought several sterile individually wrapped cotton swabs along with me to Honduras. Once in the vineyard I swabbed several grape clusters, and carefully broke the tips of the swabs off to drop them back in their little sterile packages and wrap them up.

Pre-Enrichment

Once back home, the real fun began. I filled three old White Labs yeast vials with diluted starter wort (1.015-1.020ish) and inoculated each with two cotton swabs. The idea here is to pre-enrich your culture by creating a healthy environment for the yeast and hoping they out-compete whatever else is on that swab. It's important to remember to practice aseptic technique here too, even though you're working with an environmental sample. I would hate to have gone through all this trouble to isolate California Ale yeast because I didn't sanitize my vials. Throughout this project, I used the highest proof vodka I could find in a big cheap jug at the liquor store as my sanitizer. Frequently using it to sanitize little vials made it more convenient than mixing up some Star-San. I kept my kettle full of boiling water in case I felt like I needed to rinse whatever I had just soaked in vodka, but it's not so bad to skip that at this step. A few drops of ethanol diluted into your culture can only help to select for alcohol resistant microbes such as yeast. Every couple days I loosened the caps on the vials just enough to let gas escape. Once the little vials stopped producing gas I transferred them to flasks and fed them some more starter wort.

Appearance and aroma will tell you what's growing in your cultures. Each of the three cultures I had started were growing different things, which is exactly why it's important to have more than one attempt going. One very quickly grew an enormous bright orange fungal glob (similar to a kombucha scoby) and was disposed of quicker than I could take a picture of it. Of the other two, one was producing lots of CO2 and had that characteristic bready yeast aroma. The other was not producing any gas and smelled very strongly of vinegar. I kept feeding the culture with the yeast to continue raising the alcohol concentration until I could get a sample into the lab and plate it.

Isolation Plating

There are some great resources out there with instructions for various plating techniques that I won't really go into. As an undergraduate doing personal work in the university lab I am subject to using whatever plates are left over from other various undergraduate classes. I have in past used YPGA plates with good results, but this time PCA (Plate Count Agar) is what was available and it worked fine too. So I streaked two PCA plates and two PCA + CAM (antibiotic) plates.

Yeast grew on all four plates, all with the same colony morphology, thin creamy white colonies with a dense brighter nipple in the center. Some yellow bacteria also grew on the plates without antibiotics. To make pitchable yeast from a plate, I selected a colony and used it to inoculate a tube of diluted starter wort much the same way I built up the cotton swab cultures, but with even greater care towards sanitation since this is going into beer now. 

Microscopy

Under the scope, these yeast cells are elongated and show bipolar budding which lead me to jump to the conclusion that this was a strain of Brettanomyces. I was stoked! But after the culture had started to ferment it had a very strange smell. The weird aroma persisted for weeks and weeks, like wet cheez-its and onions. I cracked open my dusty Mycology textbook and found out there are lots of yeasts out there that have bipolar budding. Considering on all four plates I streaked the yeast colonies were of the same morphology, I concluded there probably wasn't a yeast in that culture that is suitable for brewing. But this was a really fun project and it got me very excited to try again and find something useful!

Sourdough Bread with Heady Topper and Hill Farmstead Yeast

I recently spent a week in Vermont with some cheese monger friends. We went up there as a sort of reunion/vacation and to visit the birthplaces of some of our favorite cheeses. Once all our planes had landed, Eddie (yup, that Eddie) had a stroke of brilliance and checked the weather. There was a warning out for a big winter snow storm. We drove out to our cabin and found it already covered in snow - and more was falling. What happens when five dude cheese mongers/craft beer enthusiasts get snowed into a cabin together? Most of it we agreed never to speak of... but what I can share are the grand tales of overindulgence. We overindulged.

Anyway, we decided to start saving the yeast sediment from some of the funky beers we drank to use as a sourdough starter. Sediment from the following was included: Yazoo Deux Rouges, HF Biere de Norma, and Heady Topper, just to name a few. The funky dregs all went into a mason jar, along with a small ball of dough and some maple syrup to get the bugs all active again.  A couple of days in, the little culture was very active so we busted out the flower and got to work. We wrote the final bread recipe down on a journal page which may or may not have included drawings of our souls and of a shrine which may or may not have been built to hold the dough while it rose. We were snowed in.

Ultimately we just weighed the liquid yeast and subtracted its weight from the weight of hot water to be added to the flour. We let the dough rise for about 10 hours, before leaving it in the fridge overnight and kneading then baking it the following evening. As delicious as it was with some Vermont Creamery Maple Butter, it wasn't as sour as I was hoping it would be. 

Brett-ing Bottles of Yazoo's Hefeweizen

I've always loved the hefeweizen made by my hometown brewery Yazoo. But lately every time I have one I try to imagine it with a little Brett character. It's already nice and yeasty, plenty of banana and a little clove. It's just what a hefeweizen should be, but adding some barnyard funk to that mix sounds divine. I'm fairly certain it's the only year-round offering from Yazoo that is bottle conditioned, which makes me want to play around with propagating that yeast and blending in some Brett for a funky batch of homebrew.

Just for funsies I thought it would be interesting to dose a few bottles, or a six pack, of Yazoo's Hefe with some Brett, recap the bottles, and see how they taste a couple months later. The downfall to this experiment is that they are likely to over-carbonate. Seeing as they're already carbonated and the Brett will go to town munching on residual sugars and dextrins I'll just be keeping these somewhere they can explode without killing any humans or carpets, just in case.

I've got a couple liquid Brett cultures on hand to use for this, first being CMY001. I also happen to have some ECY 20 (Bug County) on a stir plate from a recent brew, propagated up from the last little bit I didn't rinse from the vial. Now, I know what you're all thinking. "The blend ratio! It'll be way off! The bacteria grow faster!" Phooey I say! Firstly, it's February in my house right now. And it feels like it. On a fast stir plate with lots of oxygen in solution at 40 degrees, I'd bet my 3-tier that the Bretts are slowly chuggin along and the bacteria are doing nothing at all. Besides, if I'm wrong I've only lost about 8 ounces of starter wort and a six pack of Hefe. Plus it could be interesting to see what the LAB contribute over the course of the year.

So I went to town adding 7-10 drops of CMY001 or ECY20 to a couple sixers of Hefe. I recapped each bottle with some old Brewer's Best caps that have been laying around since my days of brewing extract kits, labeled the six packs and tucked them away in the laundry room (which has a cement floor - just in case.) I'll be sure to update this post with tasting notes when I finally crack into these.

----UPDATES----

- 4/23/14 - round about a month later the CMY bottles have all developed the world's smallest pellicles at the liquid surface inside the bottles! Obviously when I popped the caps on these bottles to add the drops of Brett, the blanket of CO2 escaped from inside the bottle and was replaced by oxygen. The ability of Brettanomyces to scavenge oxygen free radicals is what makes it such a champion at helping beers to age properly. Although it continues to slowly ferment dextrins and increase dryness and sometimes over carbonate an aging beer, it is scavenging and utilizing oxygen to do all that. Reactive oxygen species can cause several flavor stability issues, most obviously the oxidation of ethanol to acetaldehyde, which has recently caused me to drain pour several bottles from my cellar. The formation of pellicles at the liquid surface occurs, in part, due to a larger concentration of oxygen in the air space above the liquid than dissolved within the liquid itself and helps to protect an aging/souring beer from oxidation.

CMY001

To cultivate this yeast, I was obligated to drink two delicious 100% Brett Beers from Crooked Stave. Hop Savant and St. Bretta are both fermented only with Brettanomyces, although I have seen an email circulating from Crooked Stave confirming that depending on the batch of St. Bretta, there may also be some Lacto present. Bottles were refrigerated upright for a week or so after shipping, allowed to come to room temp, opened, bottle openings were wiped with an alcohol swab to sanitize, beer was poured into glass carefully to keep sediment in bottle. A small amount of diluted starter wort (~1.020) was added to the bottles, strongly swirled, and poured into a sanitized flask to be placed on a stir plate.

Once this culture had grown large enough most of it was used to ferment the Small Fetus beer. I transferred the last 4 ounces of it to a tiny sanitized mason jar, plated it on PCA + KAM (antibiotics), and have the jar and plate banked in my beer/yeast fridge. In retrospect, I wish I didn't have to use a plate with antibiotics in the media. I'll have to plate again if I want to find out whether or not there's Lacto in that culture, but I'm currently subject to using whatever extra plates are available in the campus lab and haven't had the luxury of time to start making my own. But a boy can dream.

Only one colony morphology is seen across the entire plate, not unlike that which was exhibited by the same strain when Chad Yakobson plated it on MYPG agar.

WLP 650 Brettanomyces bruxellensis

This culture was streaked onto a PCA + KAM agar plate directly from the WLP 650 vial. Uniform colony morphology, all forming the same wrinkly brain shape that this strain also formed when plated on WLN agar pictured on the Brettanomyces Project page. However, a purple coloration is developed. 

Jordan's Graduation Stout

I've lived with the same two guys for my entire college career. We've each taken breaks, changed our majors, transferred schools, worked our butts off, and finally found what we really enjoy doing. It's been a long six years, and now the first of us has graduated. Jordan's a fan of big bold stouts, and seeing as he graduated in December that was the perfect style to make. I knew I would be serving this out of a keg at his graduation party, so in lieu of trying to fit a 10%+ ABV stout into everyone's stomachs along with all the delicious gumbo I opted to serve something a little smaller that still packed a warming punch, a Mole Stout.

I had to throw this brew together in a quick pinch, so I adopted this recipe from The Mad Fermentationist's Breakfast Stout Riff with some minor changes, mostly in the extras. I added dried habaneros, a split vanilla bean, cocoa nibs, and a cinnamon stick. This was all a few months ago, so I'm writing this post just to keep a record of this beer because it turned out really good. The heat was strong, and mostly in the back of the throat, but not unbearable. I wanted to use ancho chiles for more balanced heat/flavor, but they were out of stock at work and I had the habaneros already. The heat totally overpowered the vanilla, cinnamon was detectable, as was the cocoa, but I could have used more of each or lowered the peppers. I might also mash at a higher temp if I brew this again, although the beer didn't finish at a really low gravity it felt a little thin even with the flaked rye and might benefit from some dextrins. 

I also racked a couple liters of this into a freshly emptied itty bitty whiskey barrel. A 5 gallon Corsair barrel has been on my wish list for some time, but given that we've been moving every couple years it hasn't seemed wise to invest in one if I'm just going to have to move it while it's full. So I picked up one of the small 2 Liter Wasmund Barrels to subdue my barrel desires. This is the first beer that went in to it after the whiskey, and it came out tasting rather harsh. Next will be the Old Ale that is also destined for my Homebrew Club's barrel project, that post is soon to come.  I also plan on giving the barrel a coat of Gulf Wax to slow the evaporation, which currently happens at a frighteningly quick rate due to the crazy surface area to volume ratio.

Mole Stout

--------------

Mash Temp: 151

Estimated OG: 1.072

Estimated IBU: 42

Efficiency: 70%

Batch Size: 5.0

Boil Time: 60

Grain Bill

----------

12.0 lbs Pale Malt - 77.52%

1.25 lbs Flaked Rye - 8.07%

0.8 lbs Chocolate - 5.3%

0.45 lbs Carafa II - 2.9%

0.34 lbs Black Patent - 2.2%

0.34 lbs Caramel 120 - 2.2%

0.28 lbs Roasted Barley - 1.8%

Hops

--------

2.4 oz Williamette Pellets - 5.3%AA @ 60 mins

0.4 oz Williamette Pellets - 5.3%AA @ 30 mins

0.2 oz Williamette Pellets - 5.3%AA @ 0 mins

Yeast

--------

WLP 001

Extras

--------

1 whirlfloc @ 15 mins

0.5 tsp Yeast Nutrient @ 15 mins

3 large dried Habanero Chiles, shredded

1 split Vanilla Bean

1 Cinnamon Stick

2 oz Cocoa Nibs

Notes

---------

5.2 gallons at 1.074

Chiles, Vanilla, Cinnamon, and Nibs all put in a muslin bag and dipped in a bowl of Four Roses Bourbon. Added that to the primary after 2 weeks, left in for 7 days before kegging. Finished out at 1.021. Kegged and force carbed.

I bottled up a couple sixers of this beer straight from the keg using a DIY beer gun made from a picnic tap with a racking cane and #2 drilled stopper. I submitted a couple bottles to a home brew competition (Bluff City Extravaganza) and this beer won bronze in Category 21: Spice/Herb/Veggie Beer!

Caitlin & Eddie's Baby Brew: Part 1

A couple buddies of mine have a baby on the way, so naturally we decided to predetermine this baby's affinity for craft beer by brewing some celebratory sours. The goal was to have something ready by the time the baby is born (June-ish) and something that would age very well in the bottle so they could open one every year on the baby's birthday. Seeing as it's already February, these two goals are impossible to meet with one beer, which is a great excuse to brew two beers! Given the celebratory nature of these beers, it seemed appropriate that we make a day out of it and have a couple friends over for a tasting while brewing.

The first brew is intended to be the beer that will bottle age well, and will spend roughly a year in primary with ECY 20 which contains many strains of Brett, as well as Sach, Pedio, and Lacto. I'm hoping the complex array of Brett strains will scavenge oxygen from these bottles for years to come. When we were planning these beers we tossed around the idea of using fruit, blueberries if the baby were a boy and raspberries if a girl. Turns out she's a girl, Annabelle, so raspberries might be added to one or both of these beers closer to bottling. Each bottle will also get a nice coat of pink or red wax.

My first inclination when planning a beer that would bottle age well was to crank up the ABV as high as I could reasonably make it. But after recently doing a little cellar clearing and opening some high ABV beers that were noticeably over the hill with a few years age I opted to keep the ABV below the tolerance threshold for all the bugs involved. After tasting a few 10 year old sours that are around 5-6% ABV I'm definitely convinced that keeping active cultures in the bottle slows the rate of oxidation and increases shelf life. I also included Dextrin Malt in addition to the high mash temp to give the Brett plenty of big meals to slowly chew on.

Big Fetus: Golden Sour
--------------------------
Batch Size: 5.5 gal
Expected OG: 1.058
Expected IBU: 12.1
Efficiency: 65%
Boil Time: 60 mins
Mash: 60 mins @ 160

Grain Bill
----------
9.0 lbs Pilsen - 62.07%
2.0 lbs Wheat Malt - 13.79%
2.0 lbs Pale Malt - 13.79%
1.0 lbs Quaker Old Fashioned Oats - 6.9%
0.5 lbs Carapils (Dextrin) - 3.45%

Hops
-------
1.0 oz Hallertauer Pellets - 4.6%AA @ 40 mins.

Extras
-------
0.5 tsp Yeast Nutrients - boiled and added in primary (+14 hours)

Yeast
-------
ECY 20 Bug County

Notes
-------
-Starter prepared morning of brew day 2/18 with 8 oz. canned 1.042 wort. Strong activity by pitching time. A small amount of starter wort was added back to the ECY 20 bottle to prop the remaining bugs up. This will be refrigerated in case I feel like plating it for isolation purposes or something.

-First boil on the blichmann burner - took forever to reach boiling due to both propane tanks being low. Ended up about a quarter gallon under volume with 5.25 gallons at 1.054, meaning I also need to adjust my efficiency down a little.

-Having a tasting during the brew day proved to be a slight distraction, nearing the end of the boil I hadn't sanitized the carboy or set up the plate chiller. The wort may have waited in the boil kettle an extra 10 minutes or so, probably slightly increasing IBUs. I also forgot to add whirlfloc and the yeast nutrients, so nutrients were boiled and added to primary the next morning, when signs of fermentation had already begun.

-Carboy kept in closet at ambient temp (60)

-By Sunday 2/23 (+5 days) Sacch had fermented out all it could and bubbling totally stopped. So begins the wait for pellicle formation.