Hello, my name is Metschnikowia pulcherrima

Eureka, back to spoilage yeasts. Today, I would like to introduce another spoilage yeast called Metschnikowia pulcherrima (anamorph Candida pulcherrima): A killer yeast with applications in the wine industry, available at Lallemand’s (aka Flavia MP346) and results from one of my countless (beer) split batch experiments. I hope there is something in here for everyone. Put on your science hats, take out your pencils & notepads and start reading. Spoiler alert: you have to interpret the results yourself.

Where do I work?

M. pulcherrima can be isolated from grapes, cherries, Drosophila spp. (fruit fly), flowers and spoiled fruits [Kurtzman et al, 2011]. A yeast commonly found in nature then. If you want to know more about M. pulcherrima and its role in the wine production, go to http://wineserver.ucdavis.edu.

What about beer?

I could not find a source where M. pulcherrima is linked to beer or discussed as potential spoilage yeast thereof. This might not be that surprising since beer is, first of all, commonly not brewed with fruits and second, it’s not really the most alcohol resistant nor metabolically advanced yeast in the universe. Apparently, M. pulcherrima seems to have an alcohol tolerance of about 5% [wineserver.ucdavis.edu, 2015]. Not really high compared to brewer’s yeast with levels of around 12% (or even higher).

What is so special about me?

One very special character of M. pulcherrima makes it a very interesting yeast for the food industry: It’s a killer yeast! The killer activity is affecting blue mold (Penicillium sp.), Botrytis cinerea (gray mold) and couple of bacteria, yeasts [Kurtzman et al, 2011]. This inhibition/killer activity seems to be linked to pulcherriminic acid which is able to form insoluble compounds ultimately depleting the medium of iron ions (which are essential for the growth of other microorganisms) [Oro et al, 2014]. Scientists therefore studied efficacies of M. pulcherrima as a biocontrol agent against the molds mentioned above to eventually prolong the storage times of fruits. If you want to know more about the killer activity and the science behind it, go to PubMed and have a look at the publication from M. Sipiczki. I think this is a very nice example to show people not affiliated with science, that spending money to investigate very odd microorganisms may even result in discoveries that can have an industrial application. Or even have an impact on drug developments against pathological molds. Or put the yeast on the radar of a yeast hunter…

Where can you find me?

flaviaAs mentioned in the introduction, Lallemand sells a M. pulcherrima product called Flavia MP346. One of the few advantages to brew in Switzerland is the fact, that everything around me is about wine. And getting wine yeasts is therefore rather easy. I therefore got myself a package of said yeast and tried to investigate the impact on beer.

According to Lallemand, Flavia MP346 is a strain isolated from Chile with the speciality of α-arabinofuranosidase secretion to increase terpenes and volatile thiols to enhance the aroma of a wine. Overall increasing the aroma complexity (mainly fruit components) in the finished product. The yeast is commonly added to the must first followed by a Saccharomyces pitch 24 h later on.

To test if there is any (detectable) effect on aroma complexity in a malt based beverage like beer, I performed a simple split batch experiment. I started with a straight forward Belgian inspired recipe (3 kg Vienna malt, 3 kg Abbey malt and 0.5 kg CaraMunich 2; 24 IBU with Saazer; OG about 1.066, brewed 12. December 2014), and split the wort in two parts. One part fermented with a Saccharomyces (US-05) only, the second part with a dose of M. pulcherrima for 24 h before pitching the same Saccharomyces strain. I was really curious to try this yeast because an effect seemed very unlikely to me since the yeast can mainly ferment glucose (see below) and is incapable of fermenting maltose. Not to forget the rather low alcohol tolerance. Bottled on 12th of January 2015 (TG_control: 1.023 (5.9 ABV), TG_Metschnikowia: 1.024 (5.9 ABV)).

2015-06-17 18.30.05 First tasting performed in June, 2015 (beer approximately five month in the bottle):

Control:

Aroma: Lots of dark fruits like figs, prunes. Caramel notes as well as burnt sugar components. Very nice!

Appearance: Red-brown color, slightly cloudy, off-white head (see picture on the left). Lots of carbonation.

Flavor: Very similar to aroma. Caramel and malts. Not much else going on (yeast character or whatever).

Mouthfeel: Light body, average carbonation, malty/sweet-caramel/bitter finish. Very nicely balanced.

Overall Impression: Nice bitter:body balance. Very classical and easy to drink. Most of the character originates from choice of malts (caramalts). Not much flavor picked up from US-05 yeast (as planned).

Metschnikowia partial ferment:

Aroma: Different (compared to control): Besides the malt character (caramel, dark fruits, burnt sugar) notes of raspberries, pepper and wild funk (mostly phenolic acids). Very pleasant aroma profile.

Appearance: Red-brown color, slightly cloudy, off-white head (see picture on the left). Lots of carbonation. Similar to control.

Flavor: Similar to aroma without the fruity components (mainly caramel & malt). Finishes with a bitter overhang (balance toward bitter).

Mouthfeel: Very light body (lighter than control), higher carbonation than control (gusher),

Overall Impression: Typical Belgian dark ale with hints of fruits as well as phenolic funk in the nose. Body on the lower end resulting in an overhang of bitterness (not very well-balanced). Seems to have attenuated more than the control resulting in a low body and over-carbonated beer. In the end, I prefer the control. Although the aroma profile of the Metschnikowia beer is nice, it’s a less drinkable beer (my opinion).

So far for the plain results. Lets put them into perspective. According to Lallemand, the yeast is used in wine to promote the fruit components. And I have the feeling that I was able to pick up such an effect in my experiment as well. The beer dosed with M. pulcherrima had pronounced notes of fruits which were not present in the control. The beer had a higher attenuation level resulting in a lower body and a bitter balanced beer.

Great! I will leave the interpretation of the experiment as well as other applications of this yeast to you. The only thing that I don’t recommend is to ferment a beer with M. pulcherrima only. Simply because of its incapability to ferment the most abundant sugars in wort (just in case someone tries that, goes horribly wrong and wants to sue me for that).

Some biochemical stats about me for yeast ranchers

First some micrographs:

metschnikowia_1metschnikowia_2Yeast grows on Sabouraud agar like any other Saccharomyces yeast. I however noticed one difference: M. pulcherrima seems to be non-cycloheximide resistant (according to http://wineserver.ucdavis.edu, 2015). I plated the Lallemand product on Sabouraud agar supplemented with + 10 mg L-1 cycloheximide and I could not detect any colonies. Now some stats summarized from Kurtzman et al (2011).

Systematic name: Metschnikowia pulcherrima (anamorph Candida pulcherrima)
Synonyms: There are a lots of accepted synonyms for this yeasts. Just some examples: Torula pulcherrima, Saccharomyces pulcherrimus 
Growth on malt agar: Cell morphology: Globose to ellipsoid, 2.5 µm x 4-10 µm. Pulcherrimin (reddish-brown) pigment present & diffuses into media
Clustering: Not described
Pseudohyphae: Not described
Pellicle formation: Not described
Fermentation: Glucose: Positive
Galactose: Weak
Sucrose: Negative
Maltose: Negative
Lactose: Negative
Raffinose: Negative
Trehalose: Negative

That’s all for today. Thanks for reading.

Bibliography

Hello, my name is Hanseniaspora uvarum (aka Kloeckera apiculata)

Eureka, we are finally back to spoilage yeasts. Today, I would like to introduce another spoilage yeast called Hanseniaspora uvarum (anamorph Kloeckera apiculata). A very acid-tolerant yeast that can be found close to everywhere and is involved in various natural fermentations such as wine and even certain beer styles. Lets have a closer look at K. apiculata and some fermentations associated with this yeast.

Where do I work?

K. apiculata is present in early grape juice, cacao, coffee fermentation, malting barley, cider fermentation, spoiled figs, tomatoes, canned black cherries, can be isolated from fresh strawberries, black currants, wine grapes and various other fruits, fruit juices and fruit syrups [Pitt and Hocking, 2009, Kurtzman et al, 2011]. Furthermore, K. apiculata could be isolated from soil, fruit flies, caterpillars and even sea water (Florida USA) [Kurtzman et al, 2011]. In summary, K. apiculata is very abundant in nature. Lets have a look at some fermentations in more detail.

A study to investigate the microflora associated with wet Coffea arabica fermentation (pulped coffee is left to ferment under water) in Tanzania identified K. apiculata [Masoud et al, 2004]. The general idea is that K. apiculata, together with other yeasts, are involved in the degradation of the pulp of the coffee beans by secreting pectinases (pectin is a polysaccharide in plants found to give structural integrity). Making it possible to get the coffee bean without any pulp remainders. Another study investigating the microflora of wet fermenting Coffea arabica in Mexico could not identify K. apiculata [Avallone et al, 2001]. Same results performed on dry fermentation (pulped coffee is left to ferment exposed to air) of Coffea arabica in Brazil where the authors could not find any K. apiculata [Silva et al, 2008]. I don’t believe that K. apiculata is not present in Mexico and Brazil. I think it might be another example of the techniques used to identify yeasts may make a difference. Some techniques (especially molecular techniques such as PCR) can be more sensitive than agar plates. Furthermore, molecular techniques can pick up non-viable yeasts (as the DNA is still present) whereas one would miss these yeasts on agar plates as the yeasts are not viable (not growing) anymore.

Moving on to Irish Cider where K. apiculata is the predominant yeast in the first fermentation phase before Saccharomyces cerevisiae takes over [Morrissey et al, 2004]. In this case, the authors could identify the apples as the source for K. apiculata. Interesting to notice is the fact that the maturation phase is dominated by Brettanomyces/Dekkera (you are welcome, fellow Brett hunters).

What about beer?

Compared to some previous featured spoilage yeasts, Kloeckera apiculata can be found in beer. Very similar to the previous mentioned natural fermentations, K. apiculata is involved in a natural beer fermentation: The Belgian Lambics. After leaving the wort cool in a coolship and inoculation/mixing in tanks, the Lambic fermentation starts within a couple of days [Fig 1]. Beginning with an increase of Enterobacteriaceae and K. apiculata within the first days [van Oevelen et al, 1977]. Right before Saccharomyces sp. take over. What K. apiculata exactly contributes to the flavor composition of Lambics (and Geuze) is not really well understood. One study shows a minor impact on fatty acids and ester production where K. apiculata can increase C8, C10 and C12 fatty acids during fermentation [Spaepen M et al, 1978].

lambic_fermentation

Fig 1: Microflora evolution in Lambic fermentation taken from van Oevelen et al, 1977

Although it is commonly accepted that K. apiculata is involved in Lambic fermentations, very recent studies to investigate the microflora at Cantillon as well as an American Coolship Ale facility (Allagsh?) don’t mention K. apiculata in their result section [Bokulich et al, 2012, Spitaels F et al, 2014]. Beside all these results, one can further pose the question how K. apiculata can even get into the wort in the first place? Maybe from the wine barrels?

What is so special about me?

K. apiculata is one of the dominant yeasts in several early natural fermentation stages. Beside that, the yeast seems to have some interesting enzymes such as beta-D-glucosidase and beta-D-Xylosidase which are key enzymes to release aromatic compounds in winemaking [Kurtzman et al, 2011].

Not only are enzymes interesting but flocculation seems to be an interesting research topic as well. As previously discussed, premature flocculation can lead to premature end of a fermentation. Studies showed that K. apiculata is able to pull down a poor flocculent S. cerevisiae strain [Sosa et al, 2008]. The authors mention a possible application such as removing any natural S. cerevisiae yeasts by co-flocculation from the grape must with K. apiculata (before fermentation). Then adding a well-defined S. cerevisiae culture for the main fermentation.

Where can you find me?

In theory and taking all the various sources into consideration where one can find K. apiculata, isolating K. apiculata from various natural sources should be rather easy. Furthermore to notice, K. apiculata is not an important human pathogen although isolates exist [Kurtzman et al, 2011].

Some biochemical stats about me for yeast ranchers

Kloeckera_apiculata_001_scale.tif

Fig 2: Kloeckera apiculata (sample received from SuiGeneris)

Data summarized from Kurtzman et al (2011).

Systematic name: Hanseniaspora uvarum (anamorph Kloeckera apiculata)
Synonyms: There are a lots of accepted synonyms for this yeasts. Just some examples: Hanseniaspora apiculata, Kloeckera brevis
Growth on YM agar: Cell morphology: Apiculate, spherical to ovoid, 1.5 -5 µm x 2.5-11.5 µm [Fig 2]
Clustering: Occurring as single cells or pairs
Pseudohyphae: May be observed
Pellicle formation: Not described
Fermentation: Glucose: Positive
Galactose: Negative
Sucrose: Negative
Maltose: Negative
Lactose: Negative
Raffinose: Negative
Trehalose: Negative

Since K. apiculata is negative for maltose fermentation and actually only capable of fermenting glucose, it is very unlikely that a single K. apiculata beer fermentation would work (as mainly maltose is present in wort). K. apiculata however should work very well with Cidre and other natural fruit juices where the most dominant sugar is glucose. That’s everything I got for Kloeckera apiculata.

Bibliography

  • Avallone S, Guyot B, Brillouet JM, Olguin E, Guiraud JP (2001) Microbiological and Biochemical Study of Coffee Fermentation. Current Microbiology, Vol 42, p 252-256
  • Bokulich NA, Bamforth CW, Mills DA (2012) Brewhouse-Resident Microbiota Are Responsible for Multi-Stage Fermentation of American Coolship Ale. PLoS ONE, Vol 7(4)
  • Kurtzman CP, Fell JW, Boekhout T (2011) The Yeasts, a Taxonomic Study. Volume 1. Fifth edition. Elsevier (Link to sciencedirect)
  • Masoud W, Cesar LB, Jespersen L, Jakobsen M (2004) Yeast involved in fermentation of Coffea arabica in East Africa determined by genotyping and by direct denaturating gradient gel electrophoresis. Yeast, Vol 21(7), p 549-56
  • Morrissey WF, Davenport B, Querol A, Dobson ADW (2004) The role of indigenous yeasts in traditional Irish cider fermentations. Journal of Applied Microbiology, Vol 97, p647-655
  • Pitt JI, Hocking AD (2009) Fungi and Food Spoilage. Springer Science & Business Media
  • Silva CF, Batista LR, Abreu LM, Dias ES, Schwan RF (2008) Succession of bacterial and fungal communities during natural coffee (Coffea arabica) fermentation. Food Microbiology, Vol 25, p 951-957
  • Spaepen M, van Oevelen D, Verachtert H (1978) Fatty Acid And Esters Produced During The Spontaneous Fermentation Of Lambic And Gueuze. J. Inst. Brew, Vol 84, p 278-282
  • Spitaels F, Wieme AD, Janssens M, Aerts M, Daniel H-M, van Landscoot A, de Vuyst L, Vandamme P (2014) The Microbial Diversity of Traditional Spontaneously Fermented Lambic Beer. PLoS ONE, Vol 9(4)
  • Sosa OA, de Nadra MCM; Farias ME (2008) Behaviour of Kloeckera apiculata flocculent strain in coculture with Saccharomyces cerevisiae. Food Technology And Biotechnology, Vol 4, p 413-418
  • Van Oevelen D, Spaepen M, Timmermans P, Verachtert D (1977) Microbiological Aspects Of Spontaneous Wort Fermentation In The Production Of Lambic And Gueuze. J. Inst. Brew, Vol 83, p 356-360

Hello, my name is Kluyveromyces marxianus

Eureka, we are back to spoilage yeasts. Today, I would like to introduce another spoilage yeast called Kluyveromyces marxianus (anamorph Candida kefyr). A yeast first described by EC Hansen in 1888 and named Saccharomyces marxianus after Marx, the person who originally isolated K. marxianus from grapes [Fonseca et al, 2008].

Where do I work?

K. marxianus can be isolated from dairy products, kefir, yoghurt, fermented milk, pozol (Mexican fermented corn dough), sorghum beer, cheese, prickly pear, decaying plants and insects. And a study reveiled, that K. marxianus is even involved in coffee fermentation [Jeong H et al, 2012; Kurtzman et al, 2011; Masoud et al, 2004; Vieira-Dalode G et al, 2007].

What about beer?

I could not find a study where K. marxianus could be identified/isolated from barley based beers. Nevertheless, there is more than just barley based beers. I would like to quickly discuss another malt based beverage called gowé made in Bénin, West Africa. This beverage is available as a cooked paste which gets diluted with milk and water leading to a sweet beverage [Vieira-Dalodé et al, 2007]. Never tried gowé myself and honestly haven’t heard about it before either.

A study to identify micro-organisms involved in the production of sorghum gowé identified K. marxianus as a dominant yeast species [Vieira-Dalodé et al, 2007]. Gowé is made from malted sorghum (Sorghum bicolour) according to the work flow shown in Fig 1. The sorghum grains are cleaned and divided into two parts. 25% of the grains get soaked, drained and left for germination before sun dried. A process very similar to the malting process used to make barley malt. The malted sorghum gets milled and kneaded with water. This mixture is left for a primary fermentation. The 75% part of sorghum is left nonmalted and 15% is combined with hot water to form a slurry which is re-combined with the remaining 60% of the nonmalted sorghum and the fermenting dough to form a mixture with a temperature of about 50-60°C. This mixture is left for a secondary fermentation resulting in gowé.

gowe_production_flow

Fig 1: Production of gowé. Figure taken from Vieira-Dalodé et al, 2007

Vieira-Dalodé et al took samples during primary and secondary fermentation to identify the lactic acid bacteria and yeasts involved in the fermentation of gowé. The authors could identify K. marxianus, Pichia anomala, Candida krusei and Candida tropicalis during the fermentation. K. marxianus as well as P. anomala were present from an early stage on and the most important species during the first hours of primary fermentation. C. krusei and C. tropicalis peaked after 12 h.

What is so special about me?

K. marxianus has several biotechnological applications and is used to produce beta-galactosidase, inulinase or pectinase [Jeong et al, 2012].. Since this is yet another yeast with biotechnological applications, its genome was sequenced in 2012 by Jeong et al. The authors sequenced strain KCTC 17555 using Illumina Genome Analyzer IIx and assembled a 10.9 Mb genome allocated into eight chromosomal groups. Of 4,998 predicted proteins, 91% were also present in Kluyveromyces lactis. Furthermore, key enzymes for xylose assimilation are also present (as previously discussed in Pichia kudriavzevii) suggesting this yeast can be used for biofuel production as well [Jeong et al, 2012].

Where can you find me?

As K. marxianus is present in dairy products and many other sources, it is very likely one could pick up this yeast from these sources. The challenging part would be to identify K. marxianus from all the possibly isolated yeasts.

Some biochemical stats about me for yeast ranchers

Data summarized from Kurtzman et al (2011).

Systematic name: Kluyceromyces marxianus (anamorph Candida kefyr)
Synonyms: There are a lots of accepted synonyms for this yeasts. Just some examples: Saccharomyces marxianus, Kluyveromyces bulgaricus, Hansenula pozolis
Growth on YM agar: Cell morphology: Ellipsoidal to cylindrical, 2-6 µm x 3-11 µm
Clustering: Occurring as single cells, pairs or short chains
Pseudohyphae: Observed
Pellicle formation: May form a thin pellicle
Fermentation: Glucose: Positive
Galactose: Weak
Sucrose: Positive
Maltose: Negative
Lactose: Variable
Raffinose: Positive
Trehalose: Negative

Since K. marxianus is negative for maltose fermentation, it is very unlikely that a single K. marxianus beer fermentation would work (as mainly maltose is present in wort). So far for the theory. If anyone out there silly enough to try this yeast for a beer fermentation, please let me know. Over and out.

Bibliography

  • Fonseca GG, Heinzle E, Wittmann C, Gombert AK (2008) The yeast Kluyveromyces marxianus and its biotechnological potential. Appl Microbiol Biotechnol, Vol 79(3), p 339-54
  • Jeong H, Lee DH, Kim SH, Kim HJ, Lee K, Song JY, Kim BK, Sung BH, Park JC, Sohn JH, Koo HM, Kim JF (2012) Genome sequence of the thermotolerant yeast Kluyveromyces marxianus var. marxianus KCTC 17555. Eukaryot Cell, Vol 11(12):1584-5. doi: 10.1128/EC.00260-12, http://www.ncbi.nlm.nih.gov/pubmed/23193140
  • Kurtzman CP, Fell JW, Boekhout T (2011) The Yeasts, a Taxonomic Study. Volume 1. Fifth edition. Elsevier (Link to sciencedirect)
  • Masoud W, Cesar LB, Jespersen L, Jakobsen M (2004) Yeast involved in fermentation of Coffea arabica in East Africa determined by genotyping and by direct denaturating gradient gel electrophoresis, Yeast, Vol 21(7), p 549-56
  • Vieira-Dalode G, Jespersen L , Hounhouigan J, Moller PL, Nago CM, Jakobsen M (2007) Lactic acid bacteria and yeasts associated with gowé production from sorghum in Bénin, Journal of Applied Microbiology, Vol 103, p 342–349

Hello, my name is Torulaspora delbrueckii

Eureka, we are back to science. Today, I would like to start with a series of posts covering various spoilage yeasts. The yeast of today is widely used in food production such as bread and bakery products but has a connection to beer as well. The yeast I am talking about is called Torulaspora delbrueckii. I stumbled upon T. delbrueckii a while ago as this yeast is apparently used in the production in Bavarian Wheat beers. However, I could not find any scientific reference discussing the use of Torulaspora in beer. The only published cases of T. delbrueckii in beer cover T. delbrueckii as spoilage organism.

Where do I work?

In general, all non-Saccharomyces yeasts are considered as spoilage yeasts associated with negative traits such as introducing off-flavors, impact on clarity and different sugar preferences leading to different attenuation levels (degree of fermentation). This is now changing and lots of efforts and research is put into examining the effects of different “spoilage” yeasts in either single inoculation or in mixed fermentations along with Saccharomyces cerevisiae, the working horse of most of the beer brewers, wine makers, spirit producers and lets not forget the bakers. One other “spoilage yeast” which gets a lot of attention lately is Brettanomyces for example.

The first positive effects of Torulaspora in mixed fermentations has been initially studied in wine where the use of Torulaspora increases the complexity of the final wines [Tataridis P, van Breda V, 2013]. And yeast products with this yeast are already available.

What about beer?

The first published evidence that Torulaspora has positive effects in beer was published by Tataridis et al in 2013. The authors fermented 3.5 L of malt extract wort (OG 1.044) each with WB-06 and a strain of Torulaspora delbrueckii and compared the beers. They first noticed that T. delbrueckii was capable of metabolizing maltose the most abundant sugar in wort. However, the fermentation using T. delbrueckii took a while longer to reach terminal gravity compared to the WB-06 fermentation (157 h vs 204 h). The beer fermented with T. delbrueckii was more hazy and had a higher terminal gravity (1.012 vs 1.009). Despite the higher terminal gravity and a slower fermentation activity, the most interesting differences could be observed in the final beers. T. delbrueckii showed higher ester notes (mainly banana, rose and bubblegum) and a decreased phenolic character than WB-06. Demonstrating that T. delbrueckii might have a potential positive role in the production of wheat beers.

Now that we covered some basics about the possible advantages of the yeast, lets look at the taxonomy and biochemistry.

A quick taxonomy journey

Questions to be addressed in this chapter are:

  1. What is the closest relative yeast of T. delbrueckii?
  2. How closely related are Saccharomyces cerevisiae and Dekkera bruxellensis (aka Brettanomyces bruxellensis) to T. delbrueckii?

To address these questions, one can look at certain DNA sequences of the different yeasts and compare them in terms of how similar they are. I will try to make this very simple here. Think of a mother yeast cell from which all existing yeasts originate and evolved during time. Kind of the ur-mother-yeast-cell. Lets assign the letter A to the mother yeast cell and B to be a yeast daughter cell of A. Let me walk you through some possible examples of B and its impact on the DNA compared to A. Please notice that this is a simplified version and I am fully aware that biology is a bit more complicated than depicted in this example.

  • B is a direct ancestor of A. B is a daughter cell of A and originates from a budding/fission event of A directly creating B. In this example, the DNA of both cells are the same (I intentionally leave mutations etc aside here)
  • B is an ancestor of A but not a direct one and evolved during time thereby changing the DNA sequences in B compared to A. B is still in the lineage of A but not very close any more due to evolutionary events. There can be several billion, billion, billion daughter cells between A and B. In general, more similar DNA sequences are more likely to be closer related
  • If B is very distant of A (in terms of DNA similarities), B is classified as separate species than A. In this case, B and A cannot interbreed any more because they are too distant of each others. S. cerevisiae and Dekkera for example would be daughter cells of A but very distant and form their own species

Lets take another example, horses. Zebra, horses and donkeys look very alike but are different species. (I intentionally leave mules aside here as this these animals are hybrids of horse and donkeys). It is very likely that all these species originate from some kind of ur-horse but individually adapted to new environments forming three different, new animals. To investigate which animal is closer related to which one, one could isolate DNA from the three animals and compare them.

To address what the relationship between T. delbrueckii and S. cerevisiae and Dekkera is, one can look at the large subunit of the ribosome (LSU rRNA). The ribosome is a complex of various subunits and is responsible for the protein synthesis in the cell. Because the ribosome is a very important machinery in a cell, the changes over time on the DNA level which encode parts of the ribosome are rather low. And can therefore be used to assess relationships among different species and strains. For T. delbrueckii, the relationship between some other yeasts is depicted in Fig 1 as a phylogeny tree. The tree begins with a common ancestor and the branches represent different fates.

Phylo_tree_torulaspora

Fig 1: Phylogeny tree of Torulaspora relatives based on LSU rRNA created using Phylogeny.fr

I included additional members from the Torulaspora genus to have some close relatives of T. delbrueckii in the tree. And Saccharomyces and Dekkera to see how they end up in the phylogeny tree. One can observe that S. cerevisiae seems to be closer to Torulaspora than Dekkera.

Addressing the closest yeast relative of Torulaspora delbrueckii is a bit more complicated. First of all, it all depends on the data one uses to construct the phylogeny trees. If you for example do not include the true closest yeast relative in the dataset you will not pick it up anyway. Looking through some published phylogeny trees makes it hard to give a final answer. In one example published by Kurtzman et al (2011), the closest relative of T. delbrueckii is S. cerevisiae (like shown in Fig 1). On the other hand, another phylogeny tree published by Kurtzman et al (2011) ends up grouping Zygotorulaspora and Zygosaccharomyces closer to Torulaspora than the Saccharomyces group. In the latter case, Zygosaccharomyces mrakii and Z. rouxii end up being the closest relatives. It is therefore not possible to give a final answer here based on my investigations. However, what is obvious from the phylogeny tree shown in Fig 1, Dekkera is farther apart from Torulaspora than Saccharomyces.

Torulaspora delbrueckii has a very long list of synonyms which include a lot of different genera like Saccharomyces, Debaryomyces, Zygosaccharomyces and Torulaspora. In 1970, Kurtzman et al assigned Torulaspora and Zygosaccharomyces to Saccharomyces leaving Debaryomyces as a separate species. Five years later, van der Walt and Johannsen recreated the genus Torulaspora and incorporated all Debaryomyces species to it as well. Nine years later, Kurtzman et al accepted all four species again. This is actually not very uncommon in yeast taxonomy which is why yeast taxonomy can be very confusing and undergo lots of changes.

One reason why Saccharomyces, Debaryomyces, Zygosaccharomyces and Torulaspora make the lives of taxonomists so hard is their biochemical and phenotypical similarities and behaviour. Thus making it hard to differentiate the species. In addition, different yeasts were initially assigned to species based on morphology and biochemical properties. Nowadays, yeasts are assigned to species based on DNA. Which can lead to a lot of taxonomical changes and re-assignments of various yeasts. That’s how it is.

Beside T. delbrueckii, five other Torulaspora species exist being T. globosa, T. franciscae, T. globosa, T. maleeae, T. microellipsoides and T. pretoriensis. All other species with the exception of T. microellipsoides and T. delbrueckii are not associated with beverages. T. microellipsoides could be isolated from apple juice, tea-beer and lemonade and is a contaminant of soft drinks [Kutzman et al, 2011].

Where can you find me?

Most of the Torulaspora species and strains are isolated from soil, fermenting grapes (wine), berries, agave juice, tea-beer, apple juice, leaf of mangrove tree, moss, lemonade and tree barks [Kutzman et al, 2011]. With a bit of luck, you may find yourself some Torulaspora or you may go with the available Torulaspora delbrueckii yeast products.

Some say that Wyeast’s WY3068 Weihenstephan is a Torulaspora delbrueckii strain or contains Torulaspora delbrueckii. At least based on micrographs, its hard to tell whether WY3068 Weihenstephan is different from a typical Ale yeast (such as WY1056 American Ale) (Fig 2, 3). If anyone has rRNA seqs from WY3068, please let me know.

WY3068

Fig 2: Wyeast 3068 Weihenstephan

WY1056

Fig 3: Wyeast 1056 American Ale

Some biochemical stats about me for yeast ranchers

Below a summary of the biochemical properties of T. delbrueckii. Data is summarized from Kutzman et al (2011). One way of differentiating between S. cerevisiae and T. delbrueckii can be performed using RFLP using HinfI on amplified ITS1-5.8S-ITS2 amplicons [van Breda, 2013]. Or obviously by sequencing the ITS1-5.8S-ITS2 amplicons.

Systematic name: Torulaspora delbrueckii
Synonyms: There are a lots of accepted synonyms for this yeasts. Just some examples: Saccharomyces delbrueckii, S. rosei, S. fermentati, S. torulosus, S. chevalieri, S. vafer, S. saitoanus, S. florenzani
Growth in malt extract: Cell morphology: Spherical, ellipsoidal, 2-6 µm x 6.6 µm
Clustering: Occurring as single cells or in pairs
Pseudohyphae: None
Pellicle formation: None
Growth in malt extract: Colony morphology: After 3 days: Butyrous, dull to glistening, and tannish-white in color
Fermentation: Glucose: Positive
Galactose: Variable
Sucrose: Variable
Maltose: Variable
Lactose: Negativ
Raffinose: Variable
Trehalose: Variable

That’s all about Torulaspora delbrueckii so far. I hope this was in a way informative to you. At least keep in mind that spoilage yeasts do not inevitably have to be bad. If one can use their potential for our advantage, we can make something really unique. Have fun playing around with Torulaspora delbrueckii.

Bibliography

  • Kurtzman CP, Fell JW, Boekhout T (2011) The Yeasts, a Taxonomic Study. Volume 1. Fifth edition. Elsevier (Link to sciencedirect)
  • Tataridis P, Kanelis A, Logotetis S, Nerancis E (2013) Use of non-saccharomyces Torulaspora delbrueckii yeast strains in winemaking and brewing. Zbornik Matice srpske za prirodne nauke, Vol 124, 415-426
  • van Breda V., Jolly N, van Wyk J (2013) Characterisation of commercial and natural Torulaspora delbrueckii wine yeast strains. International Journal of Food Microbiology, 163, 80-88

Yeast banking – #5 Frozen yeasts

Eureka, today is the last post in the series about yeast banking at home (or in a lab). Please refer to the yeast basics page for links to all the other posts. The three previous methods (agar plates, isotonic sodium chloride solutions and agar slants) work all at room temperatures or colder. But not below 0°C (32°F) since the yeasts would probably die and the media (agar and sodium chloride solution) would freeze. Storing your yeasts at colder temperatures prevents some of the growth. If the yeasts do not grow during the storage time, the chances are high to have the same exact strain after you revive them. If you store your yeasts in a refrigerator your yeast can grow (even slowly) and might mutate and try to adapt to the colder temperatures. The yeasts could therefore change and maybe lose specific characteristics. This could lead to loss of flocculation or even loss of your most loved aroma profile (banana or clove aroma in wheat yeasts for example). However, such a conversion does not have to happen. It might. And this is why a lot of labs store their microorganisms or cells at lower temperatures such as -80°C (-112°F). At this low temperature no growth occurs. Even the whole metabolism of the cells arrest. The cells kind of stops entirely. You can store your cells at this temperature for nearly forever.

I am not sure how many of you out there have a -80°C freezer at home. Most of you might have a freezer at around -20°C (-4°F). And you can store your yeasts at -20°C as well. Just don’t use a freezer with thaw-cycles. The only disadvantage here is the metabolism of the yeasts might still work and some changes could occur as well. In comparison to a storage at room temperature or colder temperatures, far fewer changes can/do happen at -20°C. And this is why freezing your yeasts is as far as I know the only method to bank your yeasts over a longer period of time (years) at home.

Description of the technique

As already described, this method here is about freezing your yeasts at -20°C (-4°F) or lower/higher if you want. For this purpose you use a special media which consists of a cryoprotectant (antifreezer) such as glycerin. Please don’t use antifreezer you use for your car. If you have your storage media ready you just add some yeasts to the media and put it in your freezer and leave it there until you want to use the yeast for a future batch. Please notice, this is about banking yeasts and not yeast storage. Only small amounts of yeasts will be frozen here. Not pitchable amounts.

I would like to mention already, this is the most sophisticated method of all the four described already (agar plates, isotonic sodium chloride solution and agar slants). I do not recommend to go with this method if you haven’t tried one of the previous ones before. If you are new to yeast banking try to bank your yeast with another technique than this one before and get some experience. I recommend the isotonic yeast storage method for beginners. If you manage to revive the yeasts without an infection you might step forward to this method. If infections occur regularly, try to find the source for the infection and work on that. This method here does not work if you have troubles with your sterility and cleanliness… It just does not. In addition, the technique below is just one way to do it. I am certain there are other ways to freeze your yeasts.

Material

Fig 1: Tube filled with storage media and yeast

– Vial, tube or any other containment you can heat sterilize to store your yeast in a freezer. I use 1.5 mL reaction tubes for this purpose (Fig 1). They are small and easy to sterilize

– Food grade glycerin. Glycerin solutions work as well as long as the glycerin concentration is above 60%. I use a 85% food grade glycerin solution I bought at a local pharmacy

– Pressure cooker or any other source to heat sterilize your tubes and the food grade glycerin

– Media. I guess dried malt extract solution or even an isotonic sodium chloride solution could work as well. I use a lab media (called YPD) as a storage media. And add some ascorbic acid as well. More about my storage media later on.

– Sterile pipettes, micro pipette with sterile tips or a sterile syringes. You need sterile devices to add the storage media to your containments before freezing and get the yeast out of the containment for reviving. See “bank the yeast” description below for further information.

Preparation

First, get the freshest, purest yeast you can get. This could be from a starter or from a fresh yeast package or vial. This is very crucial. If you freeze unhealthy yeast you could risk to either loose them entirely or have problems during reviving them. Or a different outcome of a batch of beer (attenuation, taste, flocculation etc.). Please do not freeze or bank any unhealthy yeasts. And don’t expect the yeasts to come around during banking. If you have problems during a fermentation (stuck or whatever) don’t bank the yeast afterwards and hope the yeasts will be fine. They probably won’t.

What yeast sources could you use?

Fig 2: Small yeast starter with yeast at bottom

1. Yeast starter

Get yeast directly from a fresh yeast starter. Wait until no more growth occurs. Then mix up the whole yeast starter to get the yeasts back into solution. Remove some of the volume from the yeast starter and fill your pre-sterilized containments. If you want to cool down the yeast starter to let the yeasts settle to the bottom of the flask and discard as much of the yeast starter as possible (and only pitch the yeast sediment), remove the yeasts for banking before cooling down the starter. Store the filled containments for 48 h in a refrigerator. During this step the yeast build up some important molecules they need to survive and settle to the bottom (Fig 2). Remove as much of the supernatant as possible (Fig 3). This can mostly be done by just inverting the tubes, vials etc. The yeast sediment at the bottom should stay at the bottom. Just don’t turn the tubes too fast and too slow. You now should have a nice yeast sediment at the bottom (Fig 3). The volume of the yeast sediment should be below 10% of the volume of the containment. If it is a bit more or less don’t worry. However, discard some of the sediment if it is more than 20% of the volume.

Then proceed with the steps described as “bank the yeast” below.

2. Yeast package from manufacturer

Use yeast from the manufacturer directly. Make a small yeast starter and add a few mL of yeast slurry from the package or vial (Fig 2). I use glass tubes for this purpose which are filled with 4 mL of a malt extract yeast starter media (10 g of dried malt extract dissolved in 100 mL of water) and sterilized them in a pressure cooker.

Leave the starter at room temperature for 48 h. Then proceed with the steps described as “yeast starter” above. If your yeast is very fresh, you might skip the whole starter-step and bank the yeasts directly. Therefore fill your containments with the yeasts and let them settle down in a refrigerator for 48 h then discard the supernatant. Then proceed with method described as “bank the yeast” below.

3. Yeast sediment from fermenter

I would not recommend to directly bank yeasts from a slurry. At least wash them first to get rid of trub and any dead cells and do a small yeast starter. Just harvest a small amount of the sediment (like 100 mL) and wash them with sterile water for a few times until only the viable cells remain. Discard as much of the supernatant as possible. Then make a small yeast starter (100 to 200 mL), add the washed yeast cells and leave the starter at room temperature for 48 h. Then proceed with steps described as “yeast starter” above.

4. Yeast sediment from bottles

Procedure is similar to “yeast package from manufacturer” above. Make a small yeast starter and add some of the bottle sediment. Leave the starter at room temperature for 48 h. Then proceed with steps described as “yeast starter” above.

Bank the yeast

Fig 3: Tube with yeast sediment at bottom

The yeast sediment you now have in your containments should consist of very healthy and pure yeast cells (Fig 3). Now its time to add the storage media (see below) and freeze the yeasts. I add about ten times the volume of storage media for every volume of yeast. In my case I have about 0.05 to 0.1 mL of yeast sediment (Fig 3). I therefore add 0.5 mL of storage media. To add the storage media you need a sterile device such as a pipette, micro pipette with sterile tips or sterile syringes. Please pre-sterilize the storage media in a pressure cooker for 15 min if possible. Let the storage media cool down to room temperature first before proceeding. Then add the media and either gently shake the tubes, vials or use the pipette, syringe, micro pipette for a thoroughly mix. You are basically done. Just label your containments very well and put them in your freezer. Done!

Storage media:

1. Malt extract based (haven’t try this one): For 100 mL of storage media use 10 g of dried malt extract, 50 g of glycerin and fill up to 100 mL with water. Add 0.1 g ascorbic acid (aka vitamin C) if possible. The ascorbic acid helps to stabilize the membranes of the yeasts. If you have a glycerin solution for example a 85% glycerin solution calculate the amount you need as following: 50 g divided by percentage of solution (divided by 100). In this example 50 divided by 0.85 equals 58.8 g. You therefore have to add 58.8 g of your 85% glycerin media. Sterilize the storage media in a pressure cooker for 15 min if possible.

2. YPD storage media (I use this one). The recipe for this YPD-media based storage media is from the book “Yeast: The Practical Guide to Beer Fermentation” by C. White and J. Zainasheff. For 100 mL you need: 5 g YPD bouillon, 50 g of glycerin and 0.1 g ascorbic acid. Add up with water to 100 mL. Sterilize the storage media in a pressure cooker for 15 min if possible.

Storage

Put your containments in your freezer. Nothing to do more. I use a rack for my tubes to have some organizing system (Fig 4).

Fig 4: Yeast library part 1

Reanimation

1. Make yourself a yeast starter. I recommend 100 mL for the first step. Therefore dissolve 10 g of dried malt extract in 100 mL of water, add some yeast nutrients if possible and sterilize the starter for 15 min with a pressure cooker. Cool down the starter to room temperature.

2. Get your tube, vial (or whatever containment you use for yeast banking) out of your freezer and increase the temperature as fast as possible. I let the tubes warm up in my hands. Then gently mix the yeast and storage media and add the whole content to your yeast starter. I use a micro pipette for this step. Then wait a few days until signs of fermentation arise (cloudiness, white foam, yeast sediment at bottom, bubbling etc.). Wait until a yeast sediment formed at the bottom. You can either stir your yeast starter the whole time or just leave it unstirred.

3. Prepare your next yeast starter. I normally do a 1 L stirred yeast starter as a second starter here. Therefore dissolve 100 g of dried malt extract in 1000 mL of water and sterilize it. Discard the supernatant from the first yeast starter and only transfer the yeast sediment to your next 1 L yeast starter. I recommend to taste the supernatant (before discarding) to check if the starter is okay. If the starter tastes bad probably an infection occurred. If the yeast starter tastes good, congratulations!

4. Repeat the yeast starter steps until you have the amount of yeast you need. It is hard to tell how many yeast starters you need and what volume you should choose. There are way too many different way on how to bank the yeasts. The only way to tell how many yeast cells you have would be to count the cells (have a look at this post concerning this topic).

From my experience and with the amount of yeast I bank (about 0.1 mL as it can be seen in Fig 3), I need a 100 mL yeast starter first, followed by a 1 L yeast starter, followed by another 1 L yeast starter afterwards to have approximately 100E9 cells. This would be equal to the amount of yeast you get in a Wyeast’s Activator package or White Labs vial.

My experiences with this method

My procedure looks as following. As already mentioned, I use a YPD-based storage media to bank the yeasts. And I use the tubes shown in Fig 3 for banking. After discarding the supernatant after storing the tubes 48 h in the refrigerator, I add approximately 0.5 mL of the storage media to the tubes with a micro pipette and a sterile tip (Fig 5).

Fig 5: Equipment for yeast banking. YPD-based storage media (left), yeast sediment in tube (right) and micro pipette (1000 uL) with sterile tip

Then use the micro pipette to mix the yeast and the storage media. After that the tube look like shown in Fig 1. I then put the tubes in a box (shown in Fig 4) and store them in my freezer (at -20°C).

What are the advantages and disadvantages for this method compared to the others?

Advantage Disadvantage
Long term storage method Lot of equipment necessary (freezer, lab equipment etc.)
No maintenance work Contaminations not visible
Does not require a lot of space Can’t store yeast mixtures, blends
Rather complicated method

This is for sure one of the least labor intensive methods. And the only one to store your yeast over a longer period of time. On the other hand, you do need some extra equipment such as a freezer and some lab equipment (syringe or pipette or micro pipette, containments, chemicals (ascorbic acid)). I think this method is only for the people really interested in yeast banking. And I would not recommend to go with this method if you haven’t tried one of the previous ones before. Sure the long-term storage seems very advantageous. However, do not underestimate the time and equipment you need to prepare the yeast for this banking method. On the other hand, your equipment has to be very clean and mostly sterile.

As with other methods, it is not easily visible whether your yeast is infected or not by just looking at your vial, tube etc. You will know after the first yeast starter. And you can’t bank yeast blends and other mixtures with this method as well. The ratio of the different microorganisms will eventually change during the reviving. If you do want to store a blend you might have to separate the blend before…

For all of you still interested in freezing your yeasts, I would like to mention the book “Yeast: The Practical Guide to Beer Fermentation” by C. White and J. Zainasheff again. In there are further information on how to freeze your yeasts.

This is the end of the yeast banking posts. I hope I could give you some information about the topic. Please feel free to comment and ask questions if something is not clear enough. The next posts will be about some recipes, tasting notes and yeast hunting stories again. Stay tuned!

Yeast banking – #4 Agar slants

Eureka, the banking journey goes on. The introduction to yeast banking and background of this particular post can be found here. Lets proceed with technique number three, banking yeasts on agar slants. Method number one was banking yeasts on agar plates, the second one banking yeasts in sterile solutions. Banking yeasts on agar slants is very similar to the agar plate method. However, has less disadvantages than the agar plate method. Lets have a look at the method.

Description of the technique

Fig 1: Agar slants

Agar slants are basically tubes filled with agar media (Fig 1). When you prepare agar media for plates you can use the same media to prepare some slants. After the agar media cooled down the media stays gelatinous. You now can streak some yeast colonies on the agar in the tube, seal the tube and leave the tube at room temperature for some days until colonies on the agar appear. If colonies appeared, the agar slant is ready for storage. Simple as that.

Material

– Tube or any other containment containing the agar media. Make sure you chose a containment that can be tightly sealed (with a cap etc.)
– Agar media (see post about agar plate method)
– Source to get the agar slants sterile  (for example a pressure cooker)

Preparation

1. Prepare your agar media. Best would be to mix all the components for the agar media together and heat it up a bit until the agar agar is dissolved. Agar agar is not very soluble at lower temperatures and has to be dissolved at warmer temperatures. Just heat up the whole agar media (malt agar for example) until the agar agar is dissolved and then proceed to step number two.

2. Fill your tubes (or any other containment) with the freshly prepared agar media. Seal the containment and sterilize them in a pressure cooker if possible. Let the tubes sterilize for approximately 15 min. You could even submerge the tubes in boiling water for 15 min.

3. Cool down the slants. Lay the tubes sideways to get the liquid surface as shown in Fig 1. This makes it easier later on to streak some yeasts on the agar media. Wait until the agar cooled down to room temperature.

4. Store of streak. The agar slants are now ready to bank some yeast. Or can be stored at room temperature or in a refrigerator for later use.

Bank the yeast

Just pick up a yeast colony or yeast slurry with a sterile inoculating loop and streak the stuff on the agar slant. The process is very similar to streaking some agar plates. If you need further information about how to streak agar plates or slants, please have a look at the agar plate post.

Storage

Store the agar slants in the refrigerator (not in the freezer). You could even store them at a cold, dark place.

Reanimation

Please refer to the agar plate post reanimation process.

My experiences with this method

I have to mention, I do not have a lot of experience with agar slants. I just tried it once to store yeasts for some weeks and use it to ship yeasts. I therefore can’t tell you how long you can bank yeast with agar slants. However, this is a widespread method to store yeasts. Some advantages/disadvantages of this method relative to the others I described:

Advantage Disadvantage
Rather easy method Yeasts can overgrow agar media
Not a lot of equipment necessary Contaminations happen
Contaminations can be visible Can’t store yeast mixtures, blends
Yeast trading

Let me talk about the advantages first. This is a rather easy way to bank some yeasts. You just need some tubes, agar media and an inoculating loop or any other sterile gadget to get the yeasts on the agar media. Please have a look at my agar plate post to see what equipment you could use. Second, if any contaminations happen on the agar media you might see it. However, not every contamination has to be visible. And at last, you can easily trade some yeasts with other homebrewers. Just streak some yeast on an agar slant and either wait until colonies arise and then ship or ship right after streaking.

Disadvantages. Again, with agar media the yeasts still grow. Sooner or later the agar media is overcrowded with yeast colonies. This does not occur every time. And does not have to be bad after all. Its how you look at it. I do not like overcrowded agar media. I like to see single colonies… Second, because nutrients are in the agar media other yeasts, bacteria can grow as well. Contaminations can happen. Well contaminations can always happen. Independent of the yeast banking method you choose. It is therefore really, really important to keep the yeasts away from any contaminations.

Concerning agar slants as a long-term storage method. I do not have any long-term experience with this method. Please let me know if someone out there has any long-term experience with this method. I would not be surprised if you could use agar slants to store yeast for months or even years. If long-term storage (months to years) is the main goal for you to get into yeast banking, please have a look at the sterile solution method or the next post which is about a frozen yeast library.

I have to mention again, this is not a method to bank yeast mixtures or any other mixtures, blends etc. Since some growth still occurs, the ratio between the yeasts, bacteria etc. will eventually change. Even if the ratio of the mixture stays during the storage, it could change again as soon as you recover the mixture… The only way to store mixtures is to separate the individual strains and store them separately.

To summarize. Banking yeasts with agar slants is very similar to banking yeasts with agar plates. A main difference here is the lower risk of contaminations and dehydration because agar slants are tightly sealed tubes (or any other sealed containment).

The next, final post in this series is about a frozen yeast library. This is in my opinion the only way to store your yeast for a longer period of time. Stay tuned!

Yeast banking – #2 Agar plates

Eureka, today I proceed with the series about yeast banking at home (or in a lab). The introduction (previous post) about yeast banking can be found here. Lets begin with technique number one, yeast banking with agar plates.

Description of the technique

Agar plates are basically petri dishes (Fig 1) which are filled with a gelatinous media called agar media. For that you mix yourself a agar media, heat it up and sterilize it and then pour the media in the petri dishes. As the media cools down, it gets hard and dry.

Fig 1 : Petri dish (94 mm in diameter)

The agar media usually consists of nutrients for the microorganisms to grow and agar agar (or just agar) which makes the whole agar media gelatinous. Agar is very similar to gelatin which is used in cooking. More about the agar media later on.

Agar plates are very widely used in biology labs to cultivate microorganisms such as bacteria and yeasts. For that you basically distribute a liquid sample (also known as plate or streak) with your suspended microorganisms on a agar plate and wait for colonies to arise (Fig 2). The colonies are the spots you can observe. Colonies arise from one single cell (in theory) and form a colony due to the increasing amount of cells at the same spot and become visible. Meaning, a colony are a lot of cells originating from a single cell. If you plate a defined volume of your sample on a plate you can even determine the amount of microorganisms you have in your sample. Agar plates are therefore a very basic technique in microbiology.

Fig 2: Agar plate with yeast colonies

Not only can you cultivate the microorganisms but differ between some of them if you choose the right kind of agar. Meaning, some bacteria/yeast can grow on one kind of agar and others can’t. The trick here is to use the right kind of agar. You can even add further substances such as antibiotics to avoid bacterial growth on the plate, add dyes to watch for colored colonies, add pH-indicators to watch for colonies producing acids or add further substances to avoid yeast growth… the possibilities are huge.

Lets go back to the yeast banking. Agar plates can be used to bank yeast as well (what a surprise). The easiest agar for homebrewers is malt agar which is based on dry malt extract and agar agar to get the gelatinous consistency. Recipes can be found on BKYeasts blog as well as other agar media recipes. Malt agar is not the only way to go. I use Sabouraud agar for example which is a special agar to cultivate yeasts. More about Sabouraud and my agar plating technique can be found here in a very scientific post. In the end, it does not mater which kind of agar you use as long as yeasts can grow on them. The technique and material stay the same. For beginners, malt extract agar is probably the best way to go since dried malt extract is available at homebrew suppliers. A recipe for malt agar can be found on BKYeasts blog (look for malt extract/ wort agar). Don’t bother about the pH. You basically need dried malt extract, agar agar and a way to get the whole media sterile. Unfortunately, I can’t tell you about any sources for agar. In my case, agar is available at supermarkets because agar is used in baking as well.

Material

For yeast banking with agar plates you need:

– Petri dishes (available in glass or plastic)
– Source to get the agar media sterile (for example a pressure cooker)
inoculating loop (DIY works very well)
– Source to flame the inoculating loop
– Agar (aka agar agar)
– Dried malt extract (aka DME)

Concerning the Petri dishes. Petri dishes are available at Amazon for example. Either go for glass or plastic dishes.

  • Glass petri dishes can be heat sterilized in a pressure cooker or oven. Are more expensive than plastic ones. Can be re-used.
  • Plastic petri dishes are normally just used once. They usually melt during a heat sterilization step… Plastic dishes are available as sterile and non-sterile ones. Some dishes have vents to allow gas exchange. In my opinion vents are great. However not necessary.

Next thing to remember is buying petri dishes with a cover. Concerning the diameter. There are different sizes of petri dishes available. Common diameter for dishes is about 94 mm. However, you can certainly buy bigger or smaller ones if you want. I use 94 mm diameter non-sterile plastic dishes because I do a lot of platings and do not sterilize the plates before use. Don’t had a problem with contaminations so far. If you plan on doing a lot of platings, get yourself some plastic dishes.

Source to get the agar media sterile. This is a very important step to consider. Your media has to be sterile and heated up to a boil. This is important since the agar will otherwise not be dissolved properly and not gelatinize the agar media. A pressure cooker would be the best thing to have since the pressure will prevent any boil-overs.

Inoculating loops can be bought from Amazon as well or make one yourself. It is basically a metal loop… which has to be heated up before you distribute the yeast on the plate. This is done by a flame. Gas burners, ethanol burners, candles or any other flame will do the job. For further information please have a look at the Braukaiser’s post about how to make plates and slants.

Preparation

1. Make sure you have all the necessary equipment ready

2. Make some plates according to the Braukaiser’s post. I do not want to explain all the necessary steps in this blog since others have covered this topic already.

Just remember, the Braukaiser uses glass petri dishes which can be sterilized in a pressure cooker. If you work with plastic dishes you need to sterilize the media first since the plates will probably melt during the sterilization process. On the other hand, if you sterilize your glass petri dishes with the media the plates could be very moist afterwards. And water on the plate can smear your yeast colonies afterwards… You will not get nice colonies.

3. After the plates cooled down, they are ready to be streaked. Once again, one technique is described on the Braukaiser’s blog on inocculating plates and slants. For the visual people, have a look at the following YouTube video on How to Streak a plate.

4. Leave the plate(s) at a warm plate for some days until colonies appear. If you only have nice colonies such as the one shown in Fig 2, you basically mastered the agar plating technique already. Well done!

Bank the yeast

After colonies appeared, seal the plate with a tape and the plate is ready for storage. The tape prevents the plate to dry out and the introduction of any contaminations. However, don’t be too sure about the tape. Even sealed plates can get a contamination at some point.

Fig 3: Banking yeast with agar plates

Storage

Store the plate in a refrigerator at approximately 6°C (43°F). Avoid temperatures around 0°C (32°F) or below. You can even store the plates at warmer temperatures. However, in this case a lot of maintenance work will be necessary. At warmer temperatures the yeast still grow relatively fast and will sooner or later form very big colonies. Maybe even cover the whole agar plate. In this case, you need to pick a yeast colony and plate it on another plate (also known as re-streaking or re-plating). Incubate the plate again until colonies appear. Then pick another colony and plate it on another plate… You see where this is going. At cooler temperatures above freezing temperature, the yeasts will grow relatively slow. A re-streaking will be necessary at some point as well. However, the time between theses platings will be much longer compared to plates stored at ambient temperatures.

Reanimation

This is rather easy. Just pick a colony and make a small starter. More about this process in more detail again on Braukaiser’s blog about growing yeasts from a plate.

My experiences with this method

What are the advantages/disadvantages for this method as a banking tool?

Advantage Disadvantage
Rather easy method  Plates needs storage space
Not a lot of equipment necessary  Contaminations happen
Contaminations can be visible  Lot of maintenance work necessary
Agar plates can be used for other projects  Not a long-term storage method

Let me talk about the advantages first. Compared to some other techniques in yeast banking this one is rather easy to do and you do not need sophisticated equipment. In addition, contaminations in your yeast can be visible very easy. This can be seen if any other colonies (different look) appear on the plate. If there are only one kind of colonies on the plate, the chance is high your yeast is still pure. However, a contaminations does not have to be visible… Some are not visible on the plates. Therefore, contaminations can be visible. At last, you can use the agar plates for other projects such as isolating brewer’s yeast or wild yeasts from commercial beers.

Disadvantages of the agar plates yeast banking method. If you have a lot of strains to bank, keeping them all on agar plates will use a lot of storage space. And a lot of maintenance work since you have to re-plate them periodically. And every re-streaking is an opportunity for a contamination to sneak in. As already mentioned, even a taped plate can get infected at some point. For all these reasons, banking yeasts on agar plates is not a long-term storage method in my opinion.

You might ask yourself why I wrote a post about agar plates as a banking tool after all. In my opinion, if you only use limited yeast strains (lets say three different ones) banking them on agar plates might work really well. Just pick a colony from a agar plate, make a starter and re-streak another plate with the starter’s liquid. With this method you do not only have fresh yeast on a plate but the opportunity to check for any contaminations in the starter as well.

If you do want to get into yeast stuff yourself, agar plates are a very basic technique. Even if you start your own yeast library with a different technique such as banking them in sterile solutions, slants or freeze them, agar plates still can be a useful tool to cultivate your yeasts. However, you can use the other methods and not use any agar plates as well.

At the end, let me write about contaminations. Have a look at Fig 2 again. This is how a pure, healthy yeast culture should look like on an agar plate. Now have a look at the following pictures:

Fig 4: Contamination 1

This is how a bacteria infection looks like (Fig 4). Can’t tell what kind of bacteria it was. But it was a bacteria (checked with microscope).

Fig 5: Contamination 2

The next one is quite tricky (Fig 5). There is one colony which looks different… Found it? Will solve the puzzle later on… I have no idea what this was. Haven’t checked it out.

Fig 6: Contamination 3

Next about molds: The white fluffy thing in the left upper corner (Fig 6).

Fig 7: Contamination 4

Another mold (Fig 7). I do have a lot of mold contaminations. Maybe 80% of the contaminations I have are molds…

Fig 8: Contamination 5

The last picture is no contamination. This is how a dry plate looks like (Fig 8). Chance is little to retrieve your yeast from such a plate… And the contamination colony in Fig 5 is the yellowish one on the right upper corner. Finding such odd-looking colonies can be hard. And if such contaminations appear you either get rid of the yeast or try to get rid of the contamination by re-streaking a colony on a new plate and hope for the best.

Unfortunately for me, Lactobacillus does not grow on Sabouraud agar. Lactobacillus is a very common beer spoilage bacteria. Another one is Acetobacter. If there is Lactobacillus in one of my yeast sample, I could not tell by just looking at the agar plate since no Lactobacillus colonies arise. Maybe someone out there has any experience with Lactobacillus on malt agar. I want to close this huge post with a thing to remember. Keep in mind, you just see the microorganisms capable of growing on the agar media. Any microorganisms not able to grow on the agar media will stay hidden… Stay tuned! The next post will be about banking yeast in sterile solutions.