Sequencing Wild Yeast: Brewers Team Up with Scientists to Better Understand Fermentation

Feature by | Jun 2016 | Issue #113
The White Labs analytical team, including analytical lab supervisor Katie Gardner reviewing cells under microscope. | Photo courtesy of White Labs

Before co-founding the brewpub Mollusk, Cody Lee Morris brewed in a dusty 100-year-old warehouse in Seattle where he was fond of leaving buckets of wort in the nooks and crannies of the building, hoping to catch a yeast strain that might ferment his beer. A few years ago, one of those buckets started growing a mat of yeast and bacteria. A galaxy of pellicles had formed on the liquid’s surface: something sour was brewing.

Those wild microbes eventually became the foundation of an American Wild Ale released under the Epic Ales moniker. Morris aptly named it Old Warehouse and aged it in oak red wine barrels, inoculated with the warehouse strain. It’s a complex beer that borrows from a centuries-old Belgian tradition of spontaneous fermentation. It’s also a complete mystery. What was fermenting the beer? Why did its flavor change over time? “I have no idea what exactly was in it,” Morris says of the beer.

To precisely characterize what was lurking inside the barrels, Morris teamed up with Maitreya Dunham, a professor of genome sciences at the University of Washington, who had become interested in brewer’s yeast after watching her husband’s homebrew setup bubble away. A passing curiosity turned into a full-blown passion for wild yeasts when Dunham’s lab sampled the barrel of Old Warehouse and found that the wild strain was actually an entire community of microbes all working together. Using a genetic screening technique called Hi-C sequencing, Dunham found that the sample was made up of Saccharomyces, Brettanomyces, Lactobacillus bacteria, Acetobacter bacteria and a new hybrid yeast strain she’d never seen before. It was a kaleidoscope of microbes, and one was completely new to science.

“What really got me excited about this stuff is that the very first thing we sequenced, we discovered new stuff,” she says. Her results got the National Science Foundation excited, too. Dunham recently won a $680,000 grant that aims in part to “engage hobbyist and professional brewers in yeast genetics by enlisting their help to discover new hybrid yeasts.”

Projects like Dunham’s are pushing the limits of yeast science while helping brewers understand one of their most mysterious ingredients. And they’re becoming increasingly common. With sour beers all the rage, breweries—at the behest of their customers and in search of new flavors and new challenges—are intent on producing truly “native” beers that use the most hyperlocal of ingredients: namely, yeast harvested by the brewers themselves. And by employing the latest science, brewers are finally able to eliminate some of the guesswork involved in handling and brewing with these wild strains.

A few years ago, Avery Brewing Company in Boulder, Colo., ran into a little problem with its flagship IPA. Something was off with the beer’s flavor and Dan Driscoll, a molecular biologist who worked for years in biotech before joining Avery, wanted to get as specific an answer as possible. So he reached out to scientists at nearby Colorado University to sequence the yeasts they used in-house. Driscoll hoped that a precise genetic answer obtained by sequencing—reading the yeast’s DNA to reveal its molecule-by-molecule structure—could help combat what he suspected was cross-contamination.

“What we’ve done is sequenced our own in-house yeasts to look for varying genes,” Driscoll says. Rather than collect samples, plate them on petri dishes, and try to determine what is causing spoilage by a qualitative means like measuring the appearance or color of yeast, or the “old way,” as Driscoll calls it, “the idea now is actually having a molecular-based method to definitively say there are two different yeast strains there.”

In 2014, two labs at Colorado University ran Avery’s samples and performed an exhaustive analysis called Next-Generation sequencing to read out the entire genomes of the brewery’s yeast strains. What they found by combing through the genetic readout was that a gene called PAD1 was turned on for their Wit yeast and turned off for their IPA yeast. (On and off is how geneticists commonly describe the silencing or activation of genes and the traits they encode.) “When it’s intact, PAD1 results in the production of a phenol, or a chemical compound, called 4-vinylguaiacol, which is incredibly off-putting to us in our IPA,” Driscoll explains. Avery promptly destroyed the product containing 4-vinylguaiacol. Problem solved.

Thanks to Colorado University’s cutting-edge sequencing technology, which in exchange for exposure they offered for free (Next-Gen sequencing can cost $5,000 to $10,000 per run, Driscoll says), Avery was able to identify to the genetic level why its IPA tasted off. But the brewery also sequenced five other strains in their house collection. Though none of these strains are technically “wild,” one strain of Saccharomyces cerevisiae had a particularly surprising genetic makeup. “We have sequencing data for a strain that’s as close to a wild strain as we have,” says Driscoll. “And it’s not Brett.”

Driscoll was able to leverage his scientific know-how and Colorado University’s facilities to quantitatively characterize Avery’s in-house strains. But for others it won’t be quite so easy. That’s due in part to the sheer amount of data generated by whole genome sequencing: A single yeast genome has more than 5,000 genes. So if brewers don’t have a specific target in mind, like PAD1, they’re going to need some help.

To effectively leverage the technology, breweries will need experts on hand who can annotate and translate the genetic results they’re getting from sequencing, Driscoll cautions. For now, that’s out of reach to most craft brewers, which is why they’re more likely to team up with companies like White Labs for help with characterizing, storing and, if they want, sequencing their own yeast strains.

Take it to Troels
“It’s a very fun time in brewing,” says Troels Prahl, head of R&D at White Labs, a San Diego-based yeast manufacturer on which thousands of breweries small and large have come to rely. Now, a growing number of brewers are using White Labs for a different kind of service. Today, the company offers breweries an a la carte menu for storing, characterizing, purifying and sequencing their house strains. And on an almost daily basis, Prahl is helping breweries understand what these funky strains are and how they’re changing over time.

One concern for brewers is how to store a wild strain that’s performed well. If a brewery wants to bank a mixed culture, “we can store the yeast, Swiss Bank vault style,” says Prahl. White Labs can also characterize the strain and tell brewers what they have, almost as peace of mind information. “We can identify [a strain] to the species level. This is Brettanomyces lambicus, for example,” he says.

For the hardcore brewers interested in the genetics of their strain, White Labs will sequence the yeast and help interpret the results. The demand for this service and that level of granularity of data is small, says Prahl. “We’ve been sequencing to a certain extent but it’s generally not what [brewers] need.”

Wild yeast on WLN media. | Photo courtesy of White Labs

Wild yeast on WLN media. | Photo courtesy of White Labs

Interpreting Your Results In-house
Aeronaut Brewing Co. in Somerville, Mass., happens to be one of those breweries that can interpret its sequencing results in-house. This comes as no surprise, though, given that all four co-founders are scientists with degrees from M.I.T., Harvard, Rutgers and Cornell.

Co-founder Ronn Friedlander—who has a Ph.D. in bioengineering and calls himself Aeronaut’s chief scientific officer—is currently screening several wild yeast strains in a laboratory that only really came together in the last few months. That’s especially impressive, since the brewery is barely two years old. To boot, the facility didn’t cost Aeronaut a fortune: Friedlander, through his connections with scientists in the Boston area, bought a lab appliance at an auction called an autoclave that can withstand high heat and pressure to sterilize his equipment, and found a free incubator to grow his petri dishes on Craigslist. Plus, he’s hired interns to run experiments in exchange for school credit.

Drawing on his scientific expertise, Friedlander is doing some impressive characterization work at Aeronaut. After harvesting, growing and isolating several wild yeast strains, he extracts their DNA to begin the process of sequencing their genes. Then, with a few chemicals and a small centrifuge, he can extract and send the yeast DNA off to a gene sequencing company like Beckman Coulter or Genewiz that can give him a sequence to interpret with available databases—for the cost of a six-pack.

Taking the process one step further, Friedlander is using a tabletop machine no larger than a toaster to run PCR, or polymerase chain reaction, a technique that amplifies tiny amounts of DNA into thousands of copies using starter templates called primers. Essentially, primers are incredibly precise mirror images of the exact sequence you’re looking for. By designing his own primers, Friedlander can choose exactly what sequences to look for in his microorganisms, and amplify those enough to confirm the presence of a certain yeast or bacterium. If Friedlander wants to determine that he has Brett, say, he can look for yeast sequences unique to Brettanomyces; if he wants to make sure he has Pediococcus, he can design a primer that will find a sequence unique to Pedio somewhere along that bacterium’s genome.

This kind of characterization comes especially in handy for brewers trying to eliminate “spoilers,” or microbes that have invaded or cross-contaminated batches of beer. It might sound complicated, but the good news is that interested brewers don’t necessarily need Friedlander’s pedigree or equipment to perform those kinds of genetic tests.

A Dipstick Test for Wild Yeast
With sequencing technology coming down in price, brewers can now order beer spoiler kits from companies like Invisible Sentinel that use a simplified sequencing technology to detect the presence of yeasts  like Brettanomyces and Dekkera or bacteria like Pediococcus and Lactobacillus. Using a tabletop machine and a yes/no diagnostic tool that looks like an at-home pregnancy test, brewers can determine in under three hours if they have a sour bug or not. These kits don’t sequence all of the genetic material belonging to the yeast or the bacteria, but they can give brewers an idea of what kind of species they’re dealing with.

Imagine that, a dipstick test for wild yeast. But it’s important to note that while these kinds of tools may be democratizing genetic testing, they can’t provide the high-resolution genetic data that people like Friedlander, Driscoll and others are increasingly confident enough to request and interpret. For now, these out-of-the-box testing kits meet the needs of most brewers looking to get a handle on their wild yeast, help find spoiling organisms, and improve their sour beers.

Today, though, it is the most forward-looking breweries—the handful who are running in-house PCR and teaming up with scientists that use technologies like Hi-C and Next-Gen sequencing—that are helping push the limits of yeast science and improving the quality and consistency of sours, a broad style that while historically fickle, is ultimately quite rewarding.

“Now we’re seeing that there’s not just a back-to-roots movement of spontaneous brewing,” says Prahl of White Labs. “What’s unique is brewers [who] are making new world beers that are fermentation-forward using cutting-edge science to make funky yet predictable beers.”