The Making of KRISPY 2.0

Note to readers: based on positive brewer feedback, KRISPY 2.0 is now KRISPY and the "legacy" KRISPY will be phased out.

 

You know and may have brewed with our KRISPY strain from the kveik collection -  The Search for KRISPY (clean kveik yeast) – Escarpment Labs. Since its release in 2020, this strain has been popular among home and pro brewers for several reasons. First of all, it is a kveik strain so it ferments fast and at warm temperatures (20-30°C). Also, its characteristic fruity esters give it a prominent citrusy flavour and aroma above 30°C.

Beer yeasts as we know them are good at making beer but they are not perfect. Even though KRISPY is a remarkable product, it has a slight limitation in attaining full attenuation. So, we have had to blend KRISPY with a “helper” strain in order to improve its attenuation. Still, getting KRISPY to produce drier beers has been challenging. Indeed, several brewers have reached out to us saying they would like a KRISPY strain that produces lager-like beers with a lower finishing gravity. Hence, we followed up on the request of brewers and used an ancient technology that has passed the test of time referred to as adaptive laboratory evolution (ALE) to develop a new and improved KRISPY strain with a better attenuation. ALE is based on the principles of evolution and natural selection.

The science behind adaptive laboratory evolution

Evolution happens around us all the time. Nature does this by selecting target genes required for yeasts to survive specific environments and stresses. In fact, evolution of yeasts through hundreds of years of domestication has resulted in the beer yeasts that are currently used for brewing. That is also why there is a lot of diversity among beer yeasts and no two yeasts are the same. Through continuous and repeated fermentation in wort that contains an abundance of barley sugars such as maltose and maltotriose, domesticated beer yeast have over several centuries acquired genetic changes that make them very good at consuming these sugars. This has resulted in beer yeasts that have evolved a pump that efficiently lets maltose/maltotriose into the cell to be converted into energy, carbon dioxide and ethanol. This trait is not observed in yeasts such wine yeasts (adapted to grape must fermentation and sulphite) and bread yeast that have adapted to other niches. These other yeasts have adapted to their specific environments by acquiring genetic changes specific to their environments.

Here’s how adaptive laboratory evolution works - starting with a single yeast colony, the cells in the colony continuously divide to form a population of yeast cells. Evolution of the yeast cells is triggered when this yeast population is exposed to a stressful or demanding situation called a selection pressure. Continuous exposure to a specific selection pressure will result in the yeast cells having random mutations in certain genes and/or developing other genetic changes that will result in diversity of the yeast cells within the population as several yeast variants emerge.

Over time, as the selection pressure is increased, variants that have gained superior genetic changes and mutations in key genes required to deal with the selection pressure will persist, pass on the genetic changes to their offspring and then dominate the population (Figure 1). Using ALE we are able to fast track the evolution of yeast under controlled conditions in the lab in order to get the traits that we want.

Figure 1: Overview of the adaptive laboratory evolution (ALE) experiment

Figure 1: Overview of the adaptive laboratory evolution (ALE) experiment

 

Taking a page out of nature's book

We took some inspiration from nature and performed an adaptive evolution experiment in the laboratory to make KRISPY better at fermenting wort sugars. ALE is essentially a sporting event where yeast clones in a population are made to undergo strength training for several weeks or months after which winners are selected.

The most important factor in an evolution experiment is selection pressure. For our experiment, we carefully chose a selection pressure that would drive genetic changes and make KRISPY a fast, attenuative strain. Therefore, the selection pressure we used was a high gravity wort media that had an enormous amount of maltose and maltotriose in which we re-pitched the cultures almost daily for 97 days in shake flasks at 25°C. Unlike what happens at the brewery where the yeasts are made to sit in tanks and ferment for days, we used a “forced fermentation” approach where we incubated the cultures on orbital shakers to get the yeast cells to grow and ferment faster.

At the start of the experiment, we pitched the yeast in 17°P wort media (Original gravity= 1.071) which was already stressful for the yeast. Too much sugar at this gravity puts the yeast at risk of bursting open due to osmotic shock. To get around this danger, the yeast would have to adapt fast and acquire genetic changes that will enable them to quickly use up the sugars to prevent this ordeal. After 8 re-pitches when the yeasts started feeling comfortable and got a hang of how to quickly use up the malt sugars in 17°P wort media, we began to re-pitch the yeast in a higher gravity wort media (21°P). The idea was to increase the selection pressure and allow the yeast variants that had acquired more dominant genetic changes to persist in the population. After a few more generations and re-pitching, we finally increased the specific gravity of the wort to 24°P (Original gravity = 1.102). We kept the experiment going for a few more re-pitches until we got to a point where we observed a stable growth and sugar consumption. We ended the experiment at this endpoint (97 days, 62 re-pitches). At the endpoint, the yeast population was dominated by several variants with different genetic changes and abilities to use up malt sugars.

That was the end of the strength training session for the KRISPY variants in the population. The next step was the KRISPY 2.0 pre-selection contest where we isolated fast fermenting variants from the population by plating the endpoint cultures on agar plates containing maltose as the sole carbon source. After incubating the plates for 48-72 hours, we identified and isolated 15 fast-growing colonies on the plate (Figure 2). These fast-growing colonies appeared larger in size compared to the other colonies on the plate because they had acquired the ability to consume malt sugars at a faster rate.

Figure 2: Isolation of fast fermenting KRISPY variants from the yeast population. KRISPY variants that consume maltose fast appear as large colonies on the maltose agar plates.

 

We then screened the 15 fast-growing colonies in 35 mL small-scale test ferments with the original KRISPY strain as the control for comparison. For these small scale test ferments, we pitched the KRISPY variants in 10°P hopped wort and incubated them at 25°C for 7 days and we took gravity reading on days 1, 3, 5 and 7 to determine how fast the KRISPY variants were fermenting. From the test ferments, we identified three top-performing variants and we set up a final championship contest to pick out a winner out of the top three variants identified. For the championships, we scaled up the fermentation and tested the three variants in 400 mL lab-scale fermentations (10°P hopped wort) at 25°C. This allowed us to pick the best variant (out of the three) that fermented fastest. We further confirmed the attenuation of this KRISPY variant in high gravity wort (20°P) and it outperformed the original KRISPY strain (Figure 3). Also noteworthy is that the "maltotriose stall" observed in the original KRISPY strain at Day 4 (10°P hopped wort) is circumvented by the highly attenuative KRISPY variant (Figure 3).

Figure 3: The best KRISPY variant showed an improved attenuation in "normal" gravity (10°P) wort and in high gravity wort (20°P). Note that these were small-scale ferments, and homebrew/pro brew ferments typically finish much faster due to better circulation of the yeast in the fermentor

 

Lastly, we further scaled up the fermentation (2 L) with our best KRISPY variant using a standard pale ale recipe followed by some in-house sensory analysis just to be sure that the genetic changes acquired by this strain had not negatively impacted genes associated with the strain’s flavour profile. The sensory analyses were very positive. Most of the participants who took part in the sensory mentioned that beer brewed with KRISPY 2.0 was drier. Furthermore, >45% of the participants also preferred the taste and aroma of the KRISPY 2.0 beer to beer brewed with the original KRISPY strain (Figure 4).

Figure 4: Results from the in-house sensory analysis. KRISPY 2.0 strain compared to original KRISPY blend.

 

Some responses from participants who took part in the beer sensory:

“Drier and slightly less tart than sample A [KRISPY].”

“Light orchard and apricot notes, crisp and dry, very pleasant.”

“Slightly fruity taste, very crisp.

 

What are the advantages of ALE?

The advantages are numerous. First of all, with adaptive laboratory evolution, we allow nature to do the genetic heavy lifting to improve the strains. Also, yeasts obtained by this approach are not genetically modified (ie non-GMO) and can be used for brewing without any regulatory and ethical concerns. Remember that the beer yeasts we use today are a product of centuries-long evolution experiments conducted by nature and facilitated by the domestication of yeasts.

Finally, this approach can be used to improve other yeast strains for industry-relevant traits such as alcohol tolerance, tolerance to hop acids, thermotolerance, low pH tolerance, you just name it. Some of these have already been demonstrated and we are looking forward to using ALE to improve other beer yeasts in our in collection to have new traits and features you will be interested in! The making of KRISPY 2.0 is a success story of how laboratory evolution can be used to make beer yeasts better at fermenting wort sugars.

KRISPY 2.0

KRISPY is now KRISPY 2.0. We see huge advantages to the new version of KRISPY. It's a single strain, so it will be more stable when you re-pitch it. This will also help with consistently getting a clear beer. It has higher attenuation, so you can get drier beers that are more similar to what you might get with lager yeast.

Here is some feedback we have got from brewers on the new KRISPY:

*"Very drinkable lager style beer in 7.5 days. This one's a keeper!"

“KRISPY 2.0 exceeded our expectations and resulted in a clean, crisp and clear beer in less than two weeks.”*

“We have now found the ideal Kveik yeast to replace W34/70 in all our beers.”