Catching Wild Yeast at the University of Guelph

Wild microorganisms are all around us in our day-to-day lives. Some of these can be isolated and used in brewing to produce new and innovative flavours. At Escarpment Labs, we offer our laboratory expertise to the brewing industry to help brewers capture unique, terroir-specific yeast and bacteria to create unique, brewery-specific experiences.

Sample collection from the Arboretum for organism isolation. Many of us are UoGuelph grads and have fond memories of this place! 

In October 2019, our R&D team collaborated with Shane Walker and others in the University of Guelph Food Science Department to scavenge the University Arboretum and the Guelph Center for Urban Organic Farming (GCUOF) for wild microorganisms. The hope was that this microbe safari would yield new, innovative, University of Guelph-derived yeast to produce an “All Guelph” beer. The idea is to produce a beer with a Guelph brewery using Guelph water and wild yeast isolated by us (a UofG spin-off company), to promote Canada's food university. The collected samples were taken from bark, twigs, fruits and vegetables found in the Arboretum and GCUOF, and brought back to our lab for isolation and testing.

In total, 35 samples were collected from various plants. Each sample was incubated in sterile wort spiked with 4% ethanol. This  encourages the selection of alcohol-resistant microorganisms that can withstand the brewing process. These samples were incubated for 2 weeks, before further rounds of microorganism isolation began!

 Inoculated samples to begin the micro-safari hunt. Samples included jalapeno peppers, Pine Tree bark, and okra. 

After the incubation period ended and there were visible signs of fermentation, the science fun began! Samples were aseptically streak plated onto various media to screen for yeast and bacteria, with the hope being that we would find both Saccharomyces cerevisiae and Lactobacillus species. Other organisms, including Saccharomyces paradoxus, Saccharomyces eubayanus, Lachancea species, and Acetobacter species are also of interest, but require additional downstream testing as they may not attenuate as well in some cases as other strains, or may produce off-flavours. The plates were then incubated for 3-5 days. Following incubation, a mixed culture of yeast and bacteria is typically seen, so isolation of individual strains and separating these organisms to obtain pure cultures begins. This process is continued until all organisms are isolated and purified, which can take multiple rounds of plating. 

The purified strains are then subjected to PCR to help identify them. Initially, organisms suspected to be fungi are subjected to ITS testing - which is the internal transcribed spacer region between the 18s and 5.6s ribosome genes found in fungi. This gene varies based on species, so genetic sequencing allows us to identify the organisms identity for downstream testing and strain selection. Similarly, suspected bacterial samples are subjected to amplification and sequencing of a 16s ribosome sequence found in bacteria, similar to the ITS sequence in yeast. Sequencing is also performed at the UofG by the Advanced Analysis Center (AAC) and analyzed by our talented team in-house at Escarpment Labs.

PCR results to confirm ITS amplification, which are then sent for sequencing. [editor's note: we don't always run our gels this quickly!]

From this project, a wide diversity of organisms were captured as seen in the table below. Many of these organisms are not useful for brewing, but luckily we were able to capture a few usable yeasts (bolded). 

Species identified for the University of Guelph wild capture project via PCR and sequencing.

Sample taken from UofG Campus Species Identified via Sequencing
Amaranth Hanseniaspora uvarum (100%)
Eastern White Pine Tree Hanseniaspora sp. isolate (likely Uvarum) (100%)
Lychee Tomato Flower Hanseniaspora sp. isolate (100%)
Turkestan Shrub Maple Bark Hanseniaspora sp. isolate / Uncultured yeast fungus (100%)
Mountain Mogo Pine Tree Nakazawaea holstii (100%)
Cockspur Hawthorn MRS plate Lachancea waltii culture (100%), Lachancea waltii (96%) : new species?
Raspberry Bush MRS plate Hanseniaspora uvarum
Castrin White Pine Cone [Candida] railenensis isolate (100%), Torulaspora delbrueckii (99%/100%)
Eastern White Pine Bark Hanseniaspora osmophila (100%)
Eastern White Pine Cone Lachancea quebecensis (100%), Lanchancea thermotolerans (100%)
Cockspur Hawthorn Lachancea waltii (100%), Lachancea thermotolerans (100%), Lachancea quebecensis (100%)
Cockspur Tree Thorn Torulaspora delbrueckii (100%)
Lychee Tomato Hanseniaspora osmophila (100%)
Okra Saccharomyces cerevisiae (100%)
Okra Hanseniaspora uvarum (100%)
Scotts Pine Torulaspora delbrueckii (100%)
Seabock Tree Thorn Hanseniaspora uvarum (100%), Hanseniaspora opuntiae (100%)
Squash Flower Saccharomyces cerevisiae (100%)
Sugar Maple Saccharomyces paradoxus (100%)
Black Cherry Tree Gluconobacter sp./oxydans (100%)
Habanero Pepper Pantoea agglomerans (100%), Pantoea vagans (100%), Pantoea eucalypti (100%), Pantoea brenneri (100%), Pantoea conspicua (100%)
Lychee Tomato Gluconobacter cerinus (100%), Gluconobacter thailandicus (100%), Gluconobacter frateurii (100%), Gluconobacter oxydans (100%), Gluconobacter japonicus (100%)
Squash Flower Sporolactobacillus nakayamae (100%), Sporolactobacillus laevis (100%), Sporolactobacillus laevolacticus (100%), Sporolactobacillus terrae (100%)
Sugar Maple MRS Gluconobacter cerinus (100%), Gluconobacter thailandicus (100%), Gluconobacter frateurii (100%), Gluconobacter oxydans (100%), Gluconobacter japonicus (100%), Gluconobacter nephelii (100%)

 

Typically, the screening of 50+ organisms yields 2-3 usable yeast and/or bacteria strains. In this case, two Saccharomyces cerevisiae strains and one Saccharomyces paradoxus strain were found, however, no Lactobacillus was isolated. However we did find a number of acetic acid bacteria (Gluconobacter) which could be useful for alternative beverages like kombucha. We did find something called Sporolactobacillus which seems to be a lactic acid producer, which might be worth following up on at a later date.

These samples then undergo more rounds of testing, where they are subjected to test fermentations to determine if they are capable of fermenting wort sugars and producing alcohol. If they pass the test fermentations, they are then used for a small scale, bench-top batches of beer and are subjected to sensory analysis by our in-house sensory panel for flavour profiling (and we really hope they taste good!). If a strain passes sensory evaluation, the process is complete and we can begin growing up the strain for use by the brewery/organization!  

Gravity checks on the test ferments to determine the sugar consumption by the organisms. The starting gravity of the test wort was 1.035 specific gravity. 

UofG Sample TF Day 1 Check TF Day 2 Check TF Day 4 Check TF Day 7 Check TF Day 14 Check
Okra
Sacch cerevisiae
1.0293 1.0206 1.0127 1.0092 1.0084
Sugar Maple
Sacch paradoxus
1.0341 1.0340 1.0337 1.0336 1.026
Squash Flower
Sacch cerevisiae
1.0297 1.0236 1.0101 1.0068 1.0052

Both the Okra and Squash Flower yeasts performed very well in fermenting the sugars to produce beer. A final gravity of 1.000-1.010 is an expected final gravity for many beers (style dependant). However, the Sugar Maple yeast, which is a non conventional yeast (Saccharomyces paradoxus), was not able to ferment all of the sugars and struggled to produce much alcohol. From this test, only the Okra and Squash Flower yeasts will be used to produce small-scale beers to identify the flavour profile of the organism. However it is possible the S. paradoxus from sugar maple could be used for low-alcohol beer production, or for alcoholic beer production in conjunction with an amyloglucosidase enzyme. 

 The change in specific gravity over two weeks for the UofG isolated yeasts subjected to forced fermentations. 

Each strain was grown up through our standard in-house process at lab scale, and was pitched at 1 million cells/mL/degree Plato. The wort base used for the test was kept very simple to allow for expression of the yeast flavours to come through in the final product. The beer was produced using Pale Malt and Wheat Malt hopped to 28 IBU during the boil. The OG (original gravity) of the wort was 1.047 with a pH of 5.37. 

Tasting notes of finished trial beers. 

Okra (Saccharomyces cerevisiae) Squash Flower (Saccharomyces cerevisiae)
Beer Information Beer Information
FG (Final Gravity): 1.006
Finishing pH: 4.46
FG: 1.010
Finishing pH: 4.42
Yeast Flavour Profile Yeast Flavour Profile
Smell:
- Citrus (orange), pineapple and low levels of red-fruit
Taste:
- Fruity (orange), notable bitterness
- Some DMS, some floral notes, no pineapple flavour
- Would be great in an English Ale
Smell:
- Much more subtle, some green apple aroma, some citrus notes (lemon)
Taste:
- Some lemon flavour, some apple flavour, no bitterness
- Overall very neutral, could be used for any style

These yeasts can now be grown up into larger quantities and used in larger scale brews to further test their abilities. The University will be trialing these strains with their own brews to create an all University of Guelph beer.

The capture, identification and selection of yeasts suitable for brewing is a highly involved and labour- intensive process. A typical timeline for this process will vary depending upon the number of samples, but will often take between 4-7 months to complete. For example, this project started in October 2019 and pure yeast samples were provided to the University in February 2020. Even then, many of the organisms captured throughout the process were not usable for conventional beer production, and the successful yeast strains still require further testing under larger scale brewing environments. However, at the end of the process, brewers are able to bank their strains and have exclusive rights to their own yeast, with a story of terroir that relates to their brewery, which is a great marketing tool!  

Escarpment Labs will be accepting four wild capture projects starting in September/October 2020, as the fall is the best time to go and forage the forests and trees around your brewery for wild yeasts! It’s all very Pokemon-Go-esque in real life but you just can’t see what you’re catching until a few months later!