Wild Yeast Research

[xico]

I figured in the interest of keeping my research "open" I would share my progress with the community here.

The project examines the types and varieties of organisms found in natural and human environments around central Washington state. I've collected samples of soil, bark, flowers, leaves/needles, sap, fungi, moss, etc. from four distinct biomes in the county I live. I am now beginning to collect samples from an active hop farm, vineyard, and orchard as well as a wild hop area, orchard abandoned 25 years back, and fruit and oak trees around the town of Ellensburg.

All samples are split into two flasks, each fermented at different temps (8C and 20C) and incubated 3-6 weeks. The media I made is highly selective with 5% ethanol and a pH of 3.5. I'm now beginning to develop the appropriate method to isolating the cultures onto plates, to be frozen down for future examination. Early Septmember I will be sequencing all the samples to determine the varieties of organisms found in the area.

Any Saccharomyces found will be compared to a running database of thousands of strains in Bavaria in hope of shedding more light on the hybridization history of the popular lager yeast. Bringing genetic material from the Pacific northwest to the data set will help determine the genealogy of the parentage species of Saccharomyces pastorianus.

From there, I will begin selection of yeast for brewing/distilling using media (different broths and petri dish gels) and gas and liquid chromatography (GC and HPLC). Media can be made with phenolic precursors like ferulic, coumaric, cinnamic acids will help determine is the yeast is Phenolic Off Flavor positive (POF+). Media with varying IBUs made from hop extract help to determine hop tolerance. And media with varying ratios of types of sugars determines the kinds of brews are best suited to the organism. For example, some yeast prefer fructose over glucose, or maltotriose over maltose, and this information can inform how we utilize the organism for fermentation.

GC and HPLC will be used to measure the possible presence of 10 common off-flavors created by yeast to further weed out the cultures that won't make for a pleasant product. Without getting into the details, these machines can help us quantify the presence of different compounds in tiny samples of beer. It saves a lot of time to remove the yeast that put off fresh baby diaper aromas in 10mL of broth before throwing it into your hard-earned wort. Ultimately, the ~200 yeast I may find, we will possibly yield a dozen yeast with promise so developing an efficient method of selection will be paramount to repeating this process.

In tandem with this project I will begin working with a new genomic sequencer in beta development that will allow us to compare the genetic markers of strains within a species. The aim will be to further develop the debate over the working taxa of the genus Brettanomyces. From what my mentors have found, Brettanomyces lambicus is not a species and is misnamed. It is becoming apparent that it is actually a strain of Bruxelensis. There is a lot of debate over Dekkera/Brettanomyces and our hope is to bring more data to the table.

I will follow up with news and results as they are received and analyzed if there is a show of interest in hearing more. I figured I'd make myself and project available to this community which has been so generous to me over the years.

All the best!

 

[rhys333]

Sounds like an interesting experiment. Thanks for sharing.

 

[xico]

Here are a few images of the early process. Samples are collected (like at the shown hop farm in Yakima), and dropped into a growing medium (maltose, yeast nutrients and vitamins, ethanol) and incubated at low lager and ale fermentation temps in separate units.

Flasks that produce CO2 are noted and eventually make to a plate. I freeze down a sample of the 100mL broth as a reference, then pour and spread 100microliters over each plate.

Plates (petri dishes) are made with different media to identify and select distinct organisms. For example, WLN has a dye in it that many commercial Sacch. can't metabolize so the colonies turn green. Wild yeast often can, so if you have a green colony and a white colony on this plate you may have one or more of both. It also has a pH indicator in it so if a colony is producing a lot of acid the blue opaque media will become pale green and transparent. This helps to determine potential differing cultures. Other plates contain different sugars, starches, antibiotics to select for bacteria, wild yeast, and sacch.

Every distinct culture gets gets moved to another plate to ensure we have one on its own. It is then assigned a number and added to a slant and also frozen down. Photographs are taken of the organism on a slide (shape, size, budding behavior, if bacteria determine gram -or+) and on a plate it has been growing on to form a large colony called, well, a super colony. These are additional ways to determine culture consistency. None of them are definitive measures but between temp, media, and looking at morphology and growth habits it gets us close enough that genetic work can be done.

 

[GilaMinumBeer]

You getting paid to do this? Or is this considered "fun"?

 

[xico]

You getting paid to do this? Or is this considered "fun"?

This activity is part of a research project for a thesis in biology. That said, I've been working with brewing microbiology since before the program very much for the fun of it.

 

[xico]

Update for safety purposes if other people are planning on hunting for wild yeast in British Columbia, Washington, and Oregon. I found and isolated one culture of Cryptococcus but I don't know the species. This is a pathogen that can be deadly and I was alarmed to find it in a finished media. It survived and multiplied in broth with 5% ethanol, designed for sacch, which means it can live in a finished beer. These organisms are becoming established in the Pacific northwest and should be avoided at all costs.

The responsibility is on each of us to ensure the safety of ourselves and others we share our brews with. Cells are very circular compared to other wild yeast that are elongated. There are india ink stains that can be used and a caffeic acid plates can be made to help identify these yeast. This was a big find and I hope others take precautions, I've never heard of a coolship that could kill you but be aware in this part of the world. After finding this in 1 in 96 flasks I will be checking all open fermented brews I make from now on.

 

[schmeek]

Nice setup! Where do you go to school? I did something similar at UCF (central FL) just for fun, but it looks like you have a more systematic approach.

What is your end goal... or hypothesis?

What is your HPLC method like? I tried using a Thermo Acclaim OA column with a gradient H2O/ACN mobile phase and achieved good selectivity with isovaleric, butyric, acetic, and lactic acid standards using a UV-DAD detector. Unfortunately, I wasn't able to run any beer samples as I got caught up in other projects.

I'm curious if you have an extraction method set up for your beer samples as those can be a complex matrix. What type of detector are you using?

I am very interested in your work! This is great stuff.

 

[xico]

I am at Central Washington University, the goal of the project is develop the skills and method for selection of ambient yeast. The data will be an ecological canvasing of yeast in central Washington's natural biomes, orchards, hop fields, vineyards, and towns. Mostly the data is focused on four wild areas and a handful of anthropocentric spaces.

As far as HPLC, I have no idea what I'm using yet. My experience with it is extremely limited still and I was looking forward to my friend training me at his lab. It sounds like you are experienced in this endeavor. I'd like to get some feedback from you when I get closer to this step. Before this, I still have to send out the remaining cultures for sequencing, 86 yeast, 4 bacteria, and 1 contaminant yeast from plating out 96 samples on the first run so it's been slow going.

Before I run HPLC I will culture desirable yeast (identified through Sangar sequencing) and run selection assays to narrow them down to the one or two that is worth testing with mass spec. I can offer more details on that soon,, but I have to run!

 

[schmeek]

Happy to help. I'm not sure if you are trying to simply identify wild yeast and bacteria in the areas or to find ones with desirable fermentation characteristics. If it's the latter, my first recommendation would be to skip the sequencing until you know that the bug is a good one. I have experienced mostly bad fermentation characteristics from wild organisms so I wouldn't waste your time identifying something you're not going to use. Additionally, (as you probably know) you can fairly easily identify yeast and bacteria by some simple tests that don't require PCR or sequencing.

 

[xico]

Because this is partly an ecological examination of ambient yeast, it makes sense to get all sequencing done to begin a database for future collections. qPCR is easy enough to do since I have the equipment anyway, and the primers can tease out a handful of hybrids of sacch that might help when running the Sanger sequences through Blast.

Good brewing yeast is slightly secondary for this project. I will run assays with whatever sacchs, bretts, torulasporas, hanseniasporas, and pichias I identify using POF positive media, pH tolerance, attenuation, carbohydrate utilization, and sensory analysis. Based on whatever comes back of interest from these will finally go to hplc since that is the equipment I have the more restrictive access to.

 

[JmcQc]

Really interesting!!
I am organising a similar project and your proceed seems really interesting!
Did you publish some paper relate to this research?
Is it possible to have some references you are base on?
Thank you for your help!

 

[xico]

No paper published and I have samples still incubating that need isolation. From then, it will be a process of determining the fitness for the brewing process of each sample.

The research done out of TUM in Munich publish their methods and I heavily borrowed from their work here. This is a public technical university with a leading brewing research program and they make their published work available for free with all supplemental data. Many of the researchers also provide slides of presentations that have been helpful too.