Monthly Archives: May 2016

Effects of ploidy on new lager yeast hybrids

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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!).

ploidy_YSS2016

Link to the publication: http://link.springer.com/article/10.1007/s00253-016-7588-3

Link to the presentation slides: http://beer.suregork.com/wp-content/uploads/2016/04/Krogerus_YSS2016.pdf

Abstract:

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)

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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.