Brewing split batch sour beers (Sour Blend and Lachancea thermotolerans)

I’m not the biggest fan of sour beer, but I am still very interested in brewing with non-conventional microbes. I brewed a batch of sour beer using Wyeast’s Lambic Blend and some bottle dregs around 3.5 years ago. It turned out surprisingly nice, and it got complements from many of my friends who love sour beer. Together with one of these friends, I brewed up a fresh 30L batch of wort (a couple of weeks ago), which I split into two fermenters. These two sub-batches were pitched with two different cultures: The Yeast Bay’s House Sour Blend, which contains a mixture of several different yeasts and bacteria, and a pure culture of a strain of Lachancea thermotolerans, which we’ve (our lab at VTT) isolated from an oak tree here in Finland.


Lachancea thermotolerans is a very interesting yeast, because of its ability to produce lactic acid. From a brewing perspective, this means it can applied e.g. to sour beer production. This is particularly interesting, because it allows for the production of sour beer without the cross-contamination risks associated with the use of lactic acid bacteria. Lachancea thermotolerans has recently gathered some interest in the brewing science community as well, as last year there were some published studies and conference presentations on the topic.

We haven’t really characterized our isolate of L. thermotolerans yet, so I thought I’d try it out in a homebrew batch. The pre-culture was smelling promising, with a strong fruity aroma combined with a distinct lactic tartness. As I wasn’t sure how the isolate would handle high osmotic stress or high ethanol concentrations, we decide to play it safe by brewing a relatively low-gravity wort. Using a 50/50 blend of pale ale malt and wheat malt, we aimed for a specific gravity of 1.050. As my brew kettle doesn’t fit 30 liters of wort, we decided to dilute the ‘Sour Blend’ portion of the wort with water. Hence we ended up with 15 liters of 1.049 wort for the L. thermotolerans portion, and 15 liters of 1.037 wort for the ‘Sour Blend’ portion.

For hops, we went with some old Saaz hops from 2010 I found in the freezer. We only aimed for a couple of IBUs. The L. thermotolerans portion got an additional handful of Nelson Sauvin hops I also found in the freezer (from 2013, so it had definitely lost some punch) added to it after flameout. We were hoping this would complement the fruity aroma that the yeast seems to produce and add a slight bit of extra bitterness.

After 2 weeks of fermentation (starting from around 19C, rising to around 23C), I transferred the L. thermotolerans portion to a keg for carbonation. The gravity had dropped to 1.011 (for an attenuation of around 78%), suggesting the yeast is capable of using maltotriose. This is quite an important result, as maltotriose is typically the second most abundant sugar in wort. The pH of the beer had dropped to 3.48 and the beer was definitely tasting sour, suggesting that a considerable amount of lactic acid had been produced by the yeast during fermentation.

Overall I’m very happy with the flavour and the aroma. The aroma is very fruity, with hints of stonefruit and tropical fruits. In the flavor there is a nice balance between the fruity and the sour notes. The flavour is also very clean, and with this I mean that there are no strange or off-putting off-flavours. This L. thermotolerans isolate also doesn’t produce any phenolic off-flavours (POF-).

L. thermotolerans is definitely a promising candidate for sour beer production, and I’m sure we will see more acid-producing yeasts pop up in the near future.


WLP099 and maltotriose

I posted a similar post in the ‘Milk The Funk’ Facebook group recently, and thought I’d copy it over to the blog as well. This post will be about White Labs’ WLP099, which I’ve been using recently for some fermentations. What are people’s experiences with it? Reading around on various homebrew forums etc. you get the impression that this yeast is a monster that will eat through everything you throw at it. Some people are even reporting that it ferments worts down to specific gravities closer to 1.000 (just do a search for “WLP099” on Homebrewtalk). This is very much in contrast to my own experiences with. Using single cell isolates of it in test fermentations, I’ve noticed that it is unable to use maltotriose, and usually ends up with attenuations of around 70% in wort. The only other similar experience I found after googling around was (apparently with input from Mr. White himself):

This of course very much goes against the fact that this yeast could ‘super-attenuate’ plain wort (which would at least require the use of maltotriose, and possibly even longer sugars). So I guess these high attenuations can only be achieved in worts supplemented with simple sugars? Another possibility is that the yeast is actually a blend (either intentional or unintentional; more on this later below).

I did some more digging, and came across this recent study by Borneman et al.:

In it they’ve sequenced a set of mostly wine strains, but they’ve included some White Labs strains as well: one of which is WLP099. Interestingly, it was found to belong to the wine yeast clade. So the fact that it actually seems to be a wine strain, would explain the lack of maltotriose use (and would fit with it having good ethanol tolerance). This paper unfortunately doesn’t go into physiology.

Another study that probably included this strain and does go into physiology is of course the recent Gallone et al. paper:

The strain names are unfortunately encoded, but I did some more digging. Going by the fact that WLP099 is POF-, I looked at the SNPs in the sequences of its PAD1 and FDC1 genes using the reads from the Borneman et al. paper:

Cross-referencing with all the sequences from the Gallone et al. paper, I was able to find only 7 strains containing the same SNPs (Beer024, Beer033, Beer088, Spirits002, Wine001, Wine009 and Wine013); all seven of which belong to the wine clade! Of these strains one, Beer033, seems to have a familiar origin and description (from Table S1): England, Beer (Strong Ale). I think we’ve found WLP099. Anyways, looking at Figure 3 and Table S5 we see that this strain doesn’t use maltotriose! Five of the other six strains which it could be also don’t use maltotriose. Beer088 is the only strain with the same SNPs which uses maltotriose, but its origin is Germany so it’s unlikely that strain would be WLP099.

So, looking at the different independent evidence (my own ferments, the forum post with the apparent input from Chris White, and the Borneman et al. and Gallone et al. papers) it looks like WLP099 is actually a wine strain and I think we can say for sure that it doesn’t use maltotriose and thus won’t super-attenuate in wort, unless it has been supplemented with simple sugars. So regarding finishing off high-gravity ferments with this yeast, it looks like it is only useful for worts to which you add sugar. In worts made only from malt, WLP099 would likely have little to no effect when adding it as a secondary yeast (i.e. when all of the monosaccharides and most of the maltose has been consumed).

So how are peopling still getting 80+% attentuations with WLP099 as the only yeast in all-malt worts? There was some speculation in the ‘Milk The Funk’ Facebook group, that WLP099 might actually (either intentionally or unintentionally) contain two yeast strains (two independent observations were given). If the other yeast strain was a dextrin-degrading one, such as S. cerevisiae var. diastaticus, it could explain how people are seeing ‘super-attenuation’ with WLP099. These strains produce extracellular glucoamylase enzymes which break down dextrin to glucose molecules, which WLP099 could then easily ferment. But this is of course only speculation!

Review on the use of hybrid yeasts for brewing

I’m really sorry for not posting in this blog more actively. I haven’t really been homebrewing much the past year, since we moved to a new house that needed renovating. We’ve finally renovated the garage into a brewing space and home bar, so I will hopefully be posting more actively about homebrewing now. I actually even brewed two batches of beer here in the new house last weekend, and I’ll be posting an update of them along with some pictures of the new brewing space in a future post.

Besides homebrewing, I like to write about my own and other’s beer and yeast research on the blog. We have some really interesting yeast projects going on, which I hope to be able to share with you soon. In the meanwhile, we were invited to write a review article on the use of hybrid yeasts in brewing for Applied Microbiology and Biotechnology, and I’m happy to say that the article has been published online now. In it we sum up the research that has been done on the use of artificial or de novo yeast hybrids for brewing applications, and discuss what kind of benefits they have to the process. These include creating strains with improved aroma formation, fermentation rate and stress tolerance. There is also a short section on how to create these hybrids. Feel free to have a look if you are interested, the article is open access!


Here is a link to the article:


The natural interspecies Saccharomyces cerevisiae × Saccharomyces eubayanus hybrid yeast is responsible for global lager beer production and is one of the most important industrial microorganisms. Its success in the lager brewing environment is due to a combination of traits not commonly found in pure yeast species, principally low-temperature tolerance, and maltotriose utilization. Parental transgression is typical of hybrid organisms and has been exploited previously for, e.g., the production of wine yeast with beneficial properties. The parental strain S. eubayanus has only been discovered recently and newly created lager yeast strains have not yet been applied industrially. A number of reports attest to the feasibility of this approach and artificially created hybrids are likely to have a significant impact on the future of lager brewing. De novo S. cerevisiae × S. eubayanus hybrids outperform their parent strains in a number of respects, including, but not restricted to, fermentation rate, sugar utilization, stress tolerance, and aroma formation. Hybrid genome function and stability, as well as different techniques for generating hybrids and their relative merits are discussed. Hybridization not only offers the possibility of generating novel non-GM brewing yeast strains with unique properties, but is expected to aid in unraveling the complex evolutionary history of industrial lager yeast.

Effects of ploidy on new lager yeast hybrids

As I’ve mentioned previously in many past posts (e.g. here, here and here), I’m working with and researching the properties of newly created lager yeast hybrids (for my PhD project). In the linked posts, you can read about some of our initial results from the project. These mainly established the technique and showed that de novo lager hybrids can exhibit hybrid vigor over their parent strains. Since then I’ve been looking more closely at how hybrids made from the same parent strains, but with varying ploidy levels (i.e. chromosome numbers), behave in regards to fermentation performance, aroma compound production and stress tolerance. We had some very interesting results, and we saw (at least with our hybrids) that the hybrids with higher ploidy level performed better and produced more aroma-rich beer. In order to try to understand why, we sequenced the hybrids and performed transcriptional analysis on selected genes. We saw that the higher ploidy hybrids had higher copy numbers of several genes related to aroma synthesis, and these were also transcribed at higher levels during fermentation. I held a presentation about this research at the 5th International Young Scientists Symposium on Malting, Brewing and Distilling in Chico about a month ago. You can download the presentation slides below! I’m also very happy to announce that we recently had a manuscript on this work accepted for publication in Applied Microbiology and Biotechnology. ‘Ploidy influences the functional attributes of de novo lager yeast hybrids‘ was just published online, and you can find a link to the publication below as well (it is Open Access!).


Link to the publication:

Link to the presentation slides:


The genomes of hybrid organisms, such as lager yeast (Saccharomyces cerevisiae × Saccharomyces eubayanus), contain orthologous genes, the functionality and effect of which may differ depending on their origin and copy number. How the parental subgenomes in lager yeast contribute to important phenotypic traits such as fermentation performance, aroma production, and stress tolerance remains poorly understood. Here, three de novo lager yeast hybrids with different ploidy levels (allodiploid, allotriploid, and allotetraploid) were generated through hybridization techniques without genetic modification. The hybrids were characterized in fermentations of both high gravity wort (15 °P) and very high gravity wort (25 °P), which were monitored for aroma compound and sugar concentrations. The hybrid strains with higher DNA content performed better during fermentation and produced higher concentrations of flavor-active esters in both worts. The hybrid strains also outperformed both the parent strains. Genome sequencing revealed that several genes related to the formation of flavor-active esters (ATF1, ATF2¸ EHT1, EEB1, and BAT1) were present in higher copy numbers in the higher ploidy hybrid strains. A direct relationship between gene copy number and transcript level was also observed. The measured ester concentrations and transcript levels also suggest that the functionality of the S. cerevisiae– and S. eubayanus-derived gene products differs. The results contribute to our understanding of the complex molecular mechanisms that determine phenotypes in lager yeast hybrids and are expected to facilitate targeted strain development through interspecific hybridization.

Report from the 5th Young Scientists Symposium in Chico (Part 2)

It took a little longer than expected to finish the second part of my summaries of the presentations at the 5th International Young Scientists Symposium on Malting, Brewing and Distilling, but here they are. You can find the first part here. I will hopefully have time to post the final part next week. I will also be posting a summary of my own presentation on new lager yeast hybrids.

  • Screening for the brewing ability of non-Saccharomyces yeasts by Maximilian Michel

Maximilian talked about the use of non-conventional yeasts for beer production and he had screened a range of non-Saccharomyces yeasts for brewing potential. Yeast isolates were first identified with genetic fingerprinting and RT-qPCR, and then sent through an initial screening test, which included growth on various carbon sources (glucose, fructose, sucrose, maltose, maltotriose and melibiose), hop resistance (various concentrations of iso-alpha acids), ethanol tolerance (various concentrations of ethanol) and phenolic off-flavour production. Promising strains were then chosen for 2L fermentations. He had focused especially on Torulaspora delbrueckii (but he had also looked at Schizosaccharomyces pombe, Pichia anomala, Hanseniaspora uvarum, Kluyveromyces lactis and Kluyveromyces marxianus), and out of the ten strains he had fermented with at ‘larger’ scale, only one was able to use maltose (and maltotriose). That strain also produced a fruity and berry-like flavour profile. So there are definitely gems to be found in the vast range of wild yeast that are available in nature.

  • Lachancea thermotolerans in primary beer fermentations by Jen House

Jen continued on the topic of using wild yeast in beer fermentations. Her research was on the use of Lachancea thermotolerans, which is an interesting species because of its ability to produce lactic acid. Hence, there is potential to use it in pure culture fermentations for the production of sour beer. Jen had tested three different strains of various origins in wort fermentations, and found that all three were able to use maltose, but not maltotriose. The three strains also produced more lactic acid and glycerol than the S. cerevisiae control. They also seemed to have quite low O2 requirements and were resistant to iso-alpha acids up to at least 60 IBU, which makes them interesting for brewing use. The pH only dropped to around 4.2 in her experimental fermentations, which means that they will only produce a mildly tart beer and may not be suitable for sour beers (as the only microbe). Lachancea yeasts have been isolated from the bark of oak trees, so that may be a good place to start looking in case you are interested in trying to isolate your own!

  • Biodiversity of yeast and lab population isolated from Beninese African Sorghum Beer Starter by Sedjro Emile Tokpohozin

Emile has been looking at the biodiversity of Beninese sorghum beers by isolating yeasts and lactic acid bacteria from starter cultures brought from Benin. These starter cultures aren’t made from pure yeast cultures, rather a small amount of beer from the previous batch is used as a starter culture for the next. Emile had isolated (identification by ITS-PCR and MALDI-TOF-MS) a range of yeasts (e.g. Saccharomyces cerevisiae, Candida krusei, Candida ethanolica and Debaryomyces hansenii) and lactic acid bacteria (Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus helveticus, Lactobacillus brevis and Lactobacillus paracasei) from a starter culture, and he further screened these for the ability to use various carbon and nitrogen sources, as well as beta-glucosidase ability (in order to break down the cyanogenic compound dhurrin that is found in sorghum). Several possible candidate isolates were identified and these are to be used in some pilot-scale fermentations next. Again shows how much ‘wild’ microbes are out there that are potentially useful in brewing!

  • Invited Speaker: Yeast culture collections by Kyria Boundy-Mills

This talk was a bit different, as Kyria talked about the Phaff yeast culture collection (of which she is the curator of). The Phaff collection is the fourth largest in the world, and contains thousands of yeasts. Many of the deposited yeasts have not been characterized very well, so Kyria talked about the possibility of finding ‘hidden gems’ in the collection. These could have some very interesting properties and phenotypes, relevant not only to the brewing industry, but also e.g. the biofuel industry (oleaginous yeast).

  • Relationships between the speed of fermentation and levels of flavor compounds post-fermentation by Maria Josey

Maria had examined the beer aroma compounds and modelled the fermentation kinetics (using a logistic model) of 10 successive fermentations using serially repitched yeast. The 10 fermentations all behaved quite similarly, with only minor differences in fermentation rate. There also didn’t seem to be any relationship between fermentation rate and number of times the yeast was repitched. This shows that you can easily reuse your yeast for over 10 generations without any significant effects on your fermentation (as long as your hygiene practices are good). Positive linear correlations were found though between the concentrations of several aroma compounds and the maximum fermentation rate (the B parameter in the model). Faster fermentation leads to more isoamyl acetate, isobutyl acetate, ethyl hexanoate and ethyl octanoate, which of course is something that seems logical as these compounds are synthesized from metabolic intermediates.

  • Omics analysis revealed multiple stress responses of lager yeast in the process of autolysis by Jinjing Wang

Jinjing had studied the yeast responses associated with autolysis by performing proteomic and transcriptomic analysis on yeast strains with different tendencies to autolyse. She also presented various methods for the quantification of autolysis, including measuring total protein in beer, the stability of the redox potential and nucleic acid release. Using microarray analysis they had identified a range of genes that were down- and upregulated in yeast strains that showed high tendency for autolysis (e.g. RLM1 and UBC4). To confirm the roles of RLM1 and UBC4 in the autolysis process, these genes were both knocked out and overexpressed in a production strain. Overexpression of RLM1 and knocking out UBC4 led to increased autolysis. However, one must keep in mind that autolysis is a complex process that is influenced by a range of cell functions and genes.

  • Energy state model for bottling plants by Isabel Osterroth

Isabel held the only presentation in the ‘Packaging’ topic, and she talked about an energy state model which she had developed for bottling plants. Sustainability and reducing energy use, combined with the fact that bottling plant models haven’t been made before, was the driving force for creating the model. The model described the energy use of various machines in the bottling plant depending on their operational state (machines use energy even when idle). A model that was able to predict the energy use of all the separate functions in the bottling plant was successfully created, and future work will include the use of the model for optimization purposes.

  • Impact of ascorbic acid additions in mashes by Joe Williams

Joe talked about his research on supplementing ascorbic acid to the mash, and gave a virtual tour of the pilot brewery at UC Davis. The motivation for adding ascorbic acid to the mash was to increase thiol and polyphenol formation and to decrease color development in the wort. The study was very preliminary at the moment, and it will be interesting to see the final results. The pilot brewery at UC Davis was quite impressive, featuring a six-vessel 170L brewhouse and four 20L nano-breweries. I am quite jealous.

  • Optimizing hop aroma in beer dry hopped with cascade utilizing glycosidic enzymes by Kaylyn Kirkpatrick

Kaylyn talked about the use of various glycosidic enzymes in dry hopped beer in order to release glycosidically bound aroma compounds. She had tested a range of commercial Rapidase enzymes and what effect they had on the concentrations of various hop aroma compounds in a beer dry hopped with Cascade. The addition of these enzymes seems to have had quite little effect on linalool concentrations, but the concentration of geranyl acetate seems to have been enhanced with the ‘Rapidase Hoptimase’ enzyme. Their sensory panel also noticed an increase in ‘tropical fruit’-like aroma, which could be attributed to several compounds that weren’t quantified in this experiment. It seems like an interesting idea though; using e.g. Cascade in combination with a glycosidic enzyme to replicate the aroma profile of some of the modern aroma hops (e.g. Citra). Not sure how economical such a solution is though?

  • Investigating sources of variation during dry-hopping by Daniel Vollmer

Daniel talked about methods to reduce the amount of variability between replicates in dry hopping experiments. Daniel had noticed in earlier experiments that there was quite large variation between his replicates during dry hopping experiments at pilot-scale, and thus attempted to locate sources for this variation. One of the key findings was that oxygen pickup has a large (negative) effect on hop aroma intensity, and this seemed to have been one of the largest sources of variation. Other sources was the raw material (i.e. the hops cones), which for future experiments will be ground. Another interesting observation, which I mentioned already in the summary of Tom Shellhammer’s keynote lecture, was that there is huge variability in oil content within the same hop cultivar (e.g. Cascade) from different farms. Also very interesting, as I mentioned, was that there seemed to have been no correlation between oil content and aroma intensity. So there are clearly other factors that affect hop aroma intensity as well.

Report from the 5th Young Scientists Symposium in Chico (Part 1)

I apologize again for the inactivity on the blog. I haven’t been brewing much the last half a year. The wife and I bought a house in the end of last year and we’ve been renovating it since. We finally moved in a couple of weeks ago, and have started settling in. So soon I’ll be able to return to brewing again! Anyways, last week I attended the 5th International Young Scientists Symposium on Malting, Brewing and Distilling, which was arranged at Sierra Nevada’s brewery from April 21-23, 2016 in Chico, California, USA. First of all I want to thank Ken Grossman, Sierra Nevada, Charlie Bamforth and all the other organizers for a fantastic conference (especially Sierra Nevada for their generosity)! The conference featured great scientific and social program, awesome food, a relaxed atmosphere, amazing people and delicious beer! I myself presented some of the recent research we’ve been conducting on lager yeast hybrids at VTT the past year (I’ll post a link to the presentation slides soon!). To sum up, we’ve been looking at how the ploidy of new lager yeasts affect their phenotypical properties. I’ll be writing up a more detailed post on this particular research soon, as we just had a manuscript on this work accepted.


As I mentioned, there were a lot of interesting presentations during the conference! I thought I’d write some short notes / summaries of all the presentations in case you are interested. Since there were a lot of presentations, I’m splitting this post into three parts. Anyways, here is the first third of the summaries:

  • Keynote: How Craft Brewing is Transforming the Way We Think About Hops and Hop Flavor by Tom Shellhammer

Tom opened the conference with an interesting talk on the current situation of hop use in the craft industry and hop research at OSU. Craft brewers are using more and more of the global hop production, which also has shifted from being ‘bitter hop’-dominated to being ‘aroma hop’-dominated. Tom also reminded the audience that 1 IBU is not the same as 1 ppm iso-alpha acid. This is particularly relevant with heavily dry hopped beers, where oxidized alpha acids (which are bitter, but not as bitter as iso-alpha acids) can influence the IBU value. In some commercial (dry hopped) beers that had been analysed at OSU, they had observed very high levels of oxidized alpha acids. Another point that was brought up, was that the perceived bitterness gets saturated at high IBU levels (i.e. very little sensorial difference between a 80 IBU beer and a 100 IBU beer). Tom also showed a very interesting figure (which Daniel Vollmer showed again later in his presentation), showing the relationship between hop oil content in Cascade hops sourced from different farms and the hop aroma intensity in beers brewed with these hops (determined by a sensory panel). What was extremely interesting was that there seemed to be no correlation what so ever. The beer brewed with the Cascade hops with lowest oil content actually seemed to have one of the highest aroma intensities. Furthermore, many of the Cascade hops that had the highest oil contents produced beers with the lowest aroma intensities. This just shows that blindly looking at hop oil contents in hops doesn’t actually tell very much about what kind of hop aroma it will give to the beer. If I remember correctly, Tom also suggested that there was no correlation between linalool or myrcene concentrations and the hop aroma intensity either, meaning that there are other key aroma compounds responsible for hop aroma out there that still need to be identified.

  • Towards the release of a 2-row barley variety for California craft malting and brewing by Joshua Hegarty

Joshua talked about how they have attempted to breed a 2-row barley variety that would be suitable for the ‘harsh’ growing conditions in California. These include an abundance of plant pathogens and dry conditions. They had crossed different parent strains, and selected superior varieties which they had then tested in the field. The new breeding lines had shown good yields and malting quality in the field trials. Using gene mapping they had also found several regions associated with disease tolerance in barley.

  • Impact of barley varieties on malt and beer flavor by Lindsay Barr

Lindsay presented some research on the influence of barley varieties on malt and beer flavour that had been carried out at the New Belgium Brewing. Barley variety seems to have quite a big influence on both wort and beer flavour (at least according to their sensory panel). However, there didn’t seem to be any correlation between the flavours that were observed in the wort and the beer. Beer age seemed to have had a bigger impact on the beer flavour than the barley variety.

  • Selective pressurized liquid extraction of hop oil from hop cones by Katy Orr

Katy talked about some of the hop-related research that had been done at Sierra Nevada Brewing. Her background was in environmental chemistry, where she had used different extraction methods to quantify hydrocarbons from environmental samples. Here, she talked about how they had tested two different extraction methods, selective pressurized liquid extraction and Likens Nickerson distillation, to test the efficiency of their hop torpedo. Both methods seemed to have yielded quite similar results for some of the compounds that were analysed. However, the main points that were brought up were that the extracted amount does not equal the actual contents and subsequently the importance of good internal standards (that behave chemically and physically as similarly as the compound of interest as possible).

  • Pro-oxidative effects on the storage stability of German Perle and Czech Saaz pellet hops by Mark Zunkel

Mark had compared the stability of Perle and Saaz hops exposed to oxygen at room temperature during a 9 month period. The hop storage index (HSI; which measures the loss of alpha and beta acids spectrophotometrically) of Perle remained quite stable for around 4 months, after which there was a more rapid loss of the hop acids. Saaz seemed to have remained slightly more stable than Perle, but also experienced a more rapid loss in the latter half of the experiment. Unsurprisingly, both hop varieties suffered a rapid loss of hop oil in the pro-oxidative environment (50% loss of hop oil in a week). This just shows that aroma hops should be stored cold and without the presence of oxygen!

  • The effect of hopping regime, cultivar and yeast ß-glucosidase activity on terpene alcohol levels in beer by Daniel Sharp

Daniel talked about the research he had been doing on the release of hop terpenes into beer from hop glycosides. This is an interesting topic for brewers interested in hop aroma, as aroma-active compounds can potentially be released during fermentation through the hydrolysis of hop-derived glycosides in the beer. He had tested the beta-glucosidase activity of a wide range of brewing yeast strains, and then selected strains with high and low activity. Surprisingly, beta-glucosidase activity didn’t seemed to affect the maximum hydrolysis level that was achieved during fermentation (and this level was much lower than the positive control where purified enzyme was added to wort). It just took a slightly longer time to reach this level with the low activity strain. Daniel didn’t seem to see any correlation between beta-glucosidase activity and the amount of aglycones in the beer. Higher glycoside extraction was achieved with whirlpool and dry hopping compared to kettle hopping. Some varieties that seemed to be high in glycosides were Columbus, Centennial, Simcoe and Summit.

  • Creating a gin utilizing novel Scottish Botanicals: A University-Industry collaboration by Margaux Huismann

Margaux talked about her MSc project, which was carried out as a collaboration between Edinburgh Gin and ICBD. During the project, she and 3 other students had developed a gin featuring Scottish (coastal) botanicals. They went to the Scottish coast to forage for interesting botanicals, and then distilled them in lab scale to develop a recipe. The recipe was then used at larger scale at the distillery to produce a commercial product. One botanical in particular, Bladderwrack, seemed to have given off a strong ‘fishy’ aroma during distillation, and its volatile aroma compounds were analysed in more detail. We later got to try the actual gin, and it was really nice (not at all as salty or ‘fishy’ as I first was expecting). Thanks Margaux!

  • Keynote: Impact of brewing practice on yeast performance by Katherine Smart

Katherine talked about some of the research she has been doing the last 15 years. This research has been focused mainly on repitching, yeast viability, stress tolerance and petite mutants. Most interesting to me was the work on why ‘1st Generation’ yeast (i.e. yeast that have already undergone one fermentation) seem to start fermentation faster than ‘0 Generation’ yeast (i.e. yeast that come straight out of the propagator). One cause, is that G1 yeast bud faster than G0 yeast (i.e. enter the replication cycle faster) and (if I remember correctly) are able to use glucose faster from the wort. G1 yeast also seem to use less FAN from the wort, which I found interesting (less nitrogen demand or more biosynthesis?). They had also used high-throughput screening systems to isolate osmo- and ethanol tolerant strains. A quite interesting remark was that strains are seldom good at both, i.e. an osmotolerant strain is rarely ethanol tolerant as well. One good point that was made regarding these high-throughput systems is that you find what you are looking for. These isolates may have high tolerance, but may otherwise perform badly in wort or produce off-flavours.

  • Cambridge Prize Lecture: The Influence of Yeast Handling on Petite Mutant Formation by Stephen Lawrence

Congratulations to Stephen for winning the Cambridge Prize! Stephen talked about the research he had carried out, which won him the Cambridge Prize. His research was focused on petite (or respiratory-deficient) mutants in brewing, and during his presentation he also talked about various stresses the yeast are subjected to during fermentation. Petite mutants (i.e. cells with damaged mitochondrial DNA) form during fermentation as a result of fermentation stresses, and these can accumulate when yeast is repitched for several generations. These petites perform worse in several regards compared to wild type cells, so their accumulation is not desirable from a brewer’s point of view. Some interesting points that were brought up, were that older cells (i.e. cells with more budding scars) were more susceptible to petite formation and that lower mtDNA copy numbers actually didn’t increase the likelihood of petite formation (e.g. older cells tend to have more mtDNA copy numbers). This seems to suggest that the accumulation of mtDNA damage has a higher impact on petite formation than the copy numbers of mtDNA. This was a very interesting talk, and the topic still seems to be quite poorly understood. It will be interesting to follow the topic in the future.


Parts two and three will be posted during next week!

Tasting Impressions: J&L Wedding Dunkel and Pale Ale

As I mentioned in my previous blog post, I brewed two batches of beer to my friends’ wedding that took place two weeks ago. Today I thought I’d finally write some tasting notes in case someone is interested in trying the recipes. I’m slightly more happy with the Pale Ale, but I have brewed the recipe (or at least variations of it) several times. Both beers were good though! Let’s start with the Dunkel!


The beer pours with a light brown color and it is slightly hazy. The color was a bit lighter than I expected, but then again I didn’t use that much roasted malts in this. A cream colored head is formed, but it collapses quite quickly leaving drapes of lacing along the glass. The appearance is okay. I’m not really sure why it hasn’t cleared despite the 2 months it was lagering in the keg at 0C. The aroma features some light roastiness (hints of dark chocolate), dark fruits, dark malt bread, and syrup/molasses. The aroma is quite clean and promises a malt-forward flavor. The taste is similar to aroma, with a light roastiness and bready malt tones dominating. Towards the end, a slight yeasty fruitiness joins in together with some grassy hops. The finish is quite dry and lightly bitter. Unfortunately the flavour is slightly boozy/solventy as well, which hints that the fermentation was not perfect. It was fermented in a temperature controlled fridge, so perhaps I underpitched or underaerated. The body is on the light side and the carbonation level is a bit too high. It is easy to drink and quite refreshing though. All in all, I’m quite happy, but you can definitely tell that the fermentation didn’t go perfectly. I’ll have to try again next winter.


The Pale Ale was more to my taste, and it was also the beer that got most compliments at the wedding (it also ran out first). It pours with a quite clear golden-amber color (similar to Sierra Nevada’s Pale Ale). This was slightly hazier at the wedding, so it cleared up nicely during two weeks in the fridge. A fluffy white head is also formed during the pour, and it collapses slowly leaving lots of lacing along the glass. A really nice appearance! There is lots of citrus (especially grapefruit), resin and grassy herbs in the aroma. As you can guess, it is very hop forward. The aroma is otherwise very clean and promises a really tasty beer. The taste begins with a slightly sweet caramel cookie flavor, and it is quickly joined by grapefruit and ‘tropical fruit’ hop flavors. Very fruity. The finish ends in a moderate bitterness, that has a slightly grassy and herbal quality to it. Maybe from the dry hops? I remember the beer being grassier at the wedding, so maybe it has cleaned up during these two weeks. The flavor is also very clean, and you can tell the fermentation went well. The beer has a medium body and carbonation level, and is very easy to drink and really refreshing. A really nice APA that suits my taste buds perfectly. It went quickly during the wedding so it seemed like I wasn’t the only one who liked it!

The labels to the beers were designed by my lovely wife ♥

Homebrew: Wedding APA

Last weekend I attended my friends’ wedding (thanks for the great party!), to which I had brewed two batches of beer. The first was a Dunkel and the second an American Pale Ale. I brewed the APA back in November with my brew mate, but I seemed to have forgotten to post the recipe and brewday notes. As I wrote in the Dunkel post, I’ve noticed that the most popular beers during these kinds of events are balanced and easy-to-drink beers. American Pale Ales with a relatively mild bitterness seem to be especially popular, so I used the recipe of my own Wedding APA as a base for this beer.

The malt bill was kept simple, with Maris Otter, Munich and Carapils. For the hops, I used up some opened bags of American hops from the freezer. The bitterness ended up a bit on the high side, but it was still suitable for the style. For the yeast I chose my ‘house yeast’ WLP002, which flocculates well and leaves a nice flavor profile. The brewday went quite smoothly and the beer was really popular during the wedding! I will be posting tasting notes of both the beers in an upcoming post!

[beerxml recipe= metric=true cache=-1]

Creating a new ’super fruity’ yeast strain – The best of Conan and WLP644?

In this blog post I’m briefly going to summarize how I created and improved a hybrid yeast strain between Conan (isolated from a can of Heady Topper in 2013) and WLP644. I’ve used both strains in several homebrews previously, and I’ve really liked the fruity esters they produce during fermentation. While Conan produces a really nice aroma profile, it doesn’t seem to attenuate as well as say WLP001 (most probably as a result of incomplete maltotriose use). This means that it often leaves a slight sweetness in the beer, and is a bit unpredictable. With WLP644 I’m not that sure. I’m fairly sure it uses maltotriose and ferments the beer quite dry, but this observation is only based on a single homebrew I’ve done with it. It seems to grow and ferment slightly slower than most ale strains though. So, I had the idea to generate a hybrid between the strains, in order to create the ultimate ‘super fruity’ yeast strain:

  • Lots and lots of fruity esters
  • Complete maltotriose use for high attenuation
  • Bonuses are fast fermentation and moderate flocculation

Spore-to-spore mating is the traditional method of generating yeast hybrids, but I myself am more of a fan of rare mating. Also, spore-to-spore mating couldn’t really be applied here in this case because of the poor sporulation ability of Conan. To begin, I needed to ‘tag’ the parent strains with selection markers. To do this, I selected spontaneous auxotrophs of Conan (ura-) and WLP644 (lys-) on 5-FOA and α-AA agar, respectively. This means that my Conan mutant strain isn’t able to synthesize its own uracil anymore, while the WLP644 mutant strain isn’t able to synthesize its own lysine anymore. Any hybrid between these two would inherit the functional genes from the other parent, meaning the hybrid is prototrophic again, and can again synthesize its own uracil and lysine. Hybrids can thus be selected on minimal media, which doesn’t contain uracil and lysine.


The actual hybridization process is easy, all I have to do is mix cultures of both parent strains in rich media (YP-Maltose in my case), incubate for 3 days, pellet, wash and starve the yeast, and finally spread it out on minimal media agar. Any colonies appearing on the minimal media agar are most likely hybrids. The other option is that the colonies are the parent strains, which have undergone spontaneous mutations to return to being prototrophic again. In order to ensure that we in fact have hybrids, the colonies are first purified by restreaking on fresh minimal media agar, and then transferring and streaking a single colony to YPM agar. DNA is then extracted from a single colony on the YPM plate, and interdelta-PCR is performed on the DNA.

Interdelta-PCR is a technique that is usable for differentiating Saccharomyces cerevisiae strains. Below you can see the gel and the profiles that our hybrids (H1-H4) and the parent strains produce. As we can see, the hybrids contain DNA (i.e. the bands) from both parent strains, so it is confirmed that we have successfully generated a hybrid. This is especially evident in the areas I’ve marked with red asterisks on the DNA Ladder. I won’t go into more details on the mating mechanisms, but the short answer is loss of heterozygosity at the MAT locus (for more details see here). Being a result of rare-mating, this hybrid will most likely be tetraploid (assuming that both parent strains are diploid), and contain approximately the whole genome of both parent strains (as can be seen from the gel below, hybrid H2 has already lost some DNA).


To stabilize the hybrids and also generate some potentially interesting phenotypes, I utilized meiotic recombination and segregation! During ascospore formation in yeast, meiosis and chromosomal crossover takes place, during which genes are shuffled between pairs of chromosomes. First we have to sporulate our hybrid (this can be done on 1% potassium acetate), then treat the forming ascospores with a lytic enzyme (e.g. Zymolyase), and finally dissect the ascospores with a micromanipulator. I choose to do this to hybrids H1 and H2. Both strains sporulated quite poorly and the spores had low viabilities as well (34% for H1 and 8% for H2). I selected the fastest growing colonies for further characterization, and this was four segregants from hybrid H1: H1/A4, H1/C3, H1/C4, H1/H1. The characterization was done in some small-scale (35 ml) wort fermentations. This was done in order to confirm that they still are able to grow and ferment well in wort and that they produce a lot of fruity esters.


tetrad_dissectionThe four segregants that were chosen for further characterization

The small-scale fermentations were carried out in 15 °P wort (specific gravity: 1.060), because I thought this would be representative of a typical IPA wort. 35 ml of wort was added to pre-weighed and airlock-capped plastic tubes (50 ml), after which 10 million cells / ml of wort of each yeast strain was added to start the fermentations. Fermentations were carried out at 18 °C (in retrospect, this might have been a little low), and they were monitored daily through mass loss. The alcohol content of the final beer was measured with an Anton Paar DMA5000M + Alcolyzer. Our HS-GC is in heavy use at the moment in other projects, so I wasn’t able to measure the individual ester concentrations. I did sniff the beers though to get a general idea of whether the hybrids are actually fruitier than the parent strains. The fermentations were done in wort that had only been hopped in the beginning of the boil, to make sure that the majority of the beer aroma was yeast- and not hop-derived.

minifermsThe small-scale fermentation vessels

fermentationFermentation progress

abv_ph ABV% and pH of the beers

As you can see, there was considerable differences in fermentation rate between the strains. Of the parent strains, Conan started fast, but ended slow, while WLP644 was the other way around with a slow start and faster finish. In early stages of fermentation, the H1 hybrid was also doing well, but in the end it was two of the meiotic segregants, H1/A4 and H1/C4, that reached the lowest final gravities after two weeks of fermentation (1.013 and 1.009 respectively). The Conan and WLP644 parent strains reached final gravities of 1.015 and 1.017 (this would probably have dropped slightly with a couple of more days of fermentation). There was considerable variation in pH as well, as the lowest pH values were observed in the beers fermented with H1/A4 (4.39), while the highest pH values were observed in the beers fermented with H2, H1/C4 and H1/H1 (4.64, 4.61 and 4.69). The aromas of the beers were very similar, all featuring a similar fruity tone that reminded me of various stone fruits (peach and apricot). While there were some differences in intensity between the beers, I’m not sure how much I would trust just my nose. In my opinion the strongest fruity aroma was found in the beers fermented with Conan, F1/A4 and F1/H1. The weakest aroma was found in the fastest fermenting beer, i.e. F1/C4. However, as I mentioned the actual ester concentrations would need to be measured to actually draw any conclusions. All aromas were ‘clean’, with no signs of any phenolics (both parents are POF-). The WLP644 beer had a slight sulfuric note to it though. Another thing I observed during these fermentations, was that WLP644 flocculates poorly, while Conan and all the hybrids flocculated quite well.

The next step will be to actually brew and taste some beers brewed with these yeast strains, in order to see if there actually is any real world difference. For this I was planning on brewing up a 25-liter batch of APA wort (OG around 1.050, IBUs around 50, whirlpool hops, but no dry hopping), which I would then split into 5 fermentation vessels. To these I would then pitch: Conan, WLP644, H1, H1/A4 and H1/C4. I’ll return when it is time for the brewday and ultimately the tasting notes.

References and additional reading for those interested:

Krogerus K, Magalhães F, Vidgren V, Gibson B. 2015. New lager yeast strains generated by interspecific hybridization. Journal of Industrial Microbiology & Biotechnology 42: 769-778. DOI: 10.1007/s10295-015-1597-6

Steensels J, Snoek T, Meersman E, Nicolino M, Voordeckers K, Verstrepen K. 2014. Improving industrial yeast strains: exploiting natural and artificial diversity. FEMS Microbiology Reviews 38: 947-995. DOI: 10.1111/1574-6976.12073

Tasting Impressions: Schwarzbock from 2013

I found a couple of bottles of homebrewed Schwarzbock from 2013, and I decided to see how it had developed during two years in bottle. This one ended up at an ABV of 6.8% and with a final gravity of 1.020, so a bit lighter than what was originally planned. I remember this one being very tasty when fresh, so let’s see how it tastes now!schwarzbock

The beer pours black and with a fluffy tan-colored head (it looks a lot lighter in the picture below). If you hold the glass against the light, you see that the beer actually has a dark ruby color (and is crystal clear). As the head collapses, it leaves patches of lacing along the glass. A nice appearance! The aroma is really nice as well, and it features a good combination of chocolate-like roasted tones, together with dark fruits and caramel. There is a slight alcohol note in the aroma as well, even though this is only 6.8%. The flavor is mostly roasty as well, with tones of dark chocolate and ash dominating. Behind this roast, there are tones of malt bread, biscuits and caramel that balance out the flavor. There are not many hop-derived flavors, but that was expected based on the recipe and the beer age. The finish has a moderately light bitterness though. The finish is quite dry as well, which adds to the perception of a relatively light body. This is quite a difference to the sweet and strong imperial stouts. The moderate carbonation level gives the beer some more mouthfeel though. It is quite easy to drink, and it has aged very well! Overall, a very nice beer that I would definitely brew again!