Hello, my name is Pichia kudriavzevii

Eureka, we are back to spoilage yeasts. Today, I would like to introduce another spoilage yeast called Pichia kudriavzevii (anamorph Candida krusei). This yeast was first described by V.I. Kudryavtsev in 1960 as Issatchenkia orientalis but was classified as P. orientalis in 1964 and to P. kudriavzevii in 1965. P. kudriavzevii is a very abundant yeast in the environment and can be found in soil, fruits and various fermented beverages. So far, P. kudriavzevii is mainly associated with food spoilage to cause surface biofilms in low pH products [Kurtzman, 2011].

Where do I work?

Since this yeast is very abundant, P. kudriavzevii could be isolated from various sources: fruit juice, berries, sourdough, butter-like products, culture of Tanzanian fermented togw, African fermented cassava lafu, Ghanaian fermented cocoa bean heap, sake, champagne, ginger beer, tea beer, baker’s yeast, chicken egg, human heart blood, sputum, swine waste and human feces [Kurtzman, 2011; Chan 2012]. Well this does not sound very appetizing. And in fact, this strain is not really appetizing at all. Not only are various isolates from humans and animals of P. kudriavzevii available but many clinical cases support the idea that P. kudriavzevii (Candida krusei) is a species of clinical importance. In fact, Candida krusei is the 5th most common cause of candidemia [Kurtzman, 2011]. A kind of fungi infection which can affect immunocompromised patients (such as AIDS patients). Since we are already speaking of Candida, Candida albicans another species within the Candida family is one of the most pathogenic yeasts around and leads to Candidiasis. If you want to know how the symptoms of these medical conditions look like, feel free to google. But be warned, its nasty!

Back to the possible applications of P. kudriavzevii. As I already mentioned, P. kudriavzevii can be isolated from various sources and has been found in sourdough and further research needs to be performed to elucidate its possible function [Meroth 2003; De Vuyst, 2005]. Beside sourdough, P. kudriavzevii accounted for about 30% of the isolated yeasts in a cocoa bean heap fermentation and might be involved in the citrate assimilation during the fermentation [Daniel, 2009].

What about beer?

One existing association to beer is CBS strain 5148 which was isolated from a beer wort. Beside the “traditional” beer, one publication covers the isolation of P. kudriavzevii from tchapalo, a sorghum beer brewed in Côte d’Ivoire [N’guessan, 2011]. Whether P. kudriavzevii has any benefits for the production of beer like many other non-Saccharomyces strains has yet to be determined.

What is so special about me?

What is quite remarkable in my opinion is the fact that a draft genome assembly exists for a P. kudriavzevii strain (M12). Meaning the genome of this yeast has been sequenced and the information of the DNA sequences are known, published and available to everyone. Since DNA sequencing and the assembling process requires some financial investments as well as time (been there), there have to be certain reason(s) why such a research project is initiated and funded. And that’s where it gets interesting. Apparently, P. kudriavzevii contains some neat enzymes which make this strain very interesting in biotechnology for processes such as the production of bioethanol and phytases used to increase the uptake of phosphorus by plants (biofertilizer) [Chan, 2012]. Below is a simplified pathway of the Xylose pathway including the three enzymes found in P. kudriavzevii. Xylose by the way is a sugar molecule found in wood and not that many yeasts are capable of metabolizing this kind of sugar.

Xylulose-5-phosphate can then be input into the pentose phosphate pathway (PPP) to be converted into fructose 6-phosphate which can be passed to the glycolysis pathway. Under the right conditions, one might use P. kudriavzevii strain M12 to form alcohol from wood which is a very neat way of producing bioethanol.

Where can you find me?

Don’t know any commercial sources for P. kudriavzevii. This is for sure not a bad thing as I don’t want to have people playing around with possible infectious yeasts at home anyway…

Some biochemical stats about me for yeast ranchers

Below a summary of the biochemical properties of P. kudriavzevii. Data is summarized from Kurtzman et al (2011). Since this yeast can mainly ferment glucose, its spectra of sugars is very limited. For sure not suitable to ferment an entire batch of beer wort which contains a lot of maltose which cannot be utilized by P. kudriavzevii.

Systematic name:	Pichia kudriavzevii (anamorph Candida krusei)
Synonyms:	There are a lots of accepted synonyms for this yeasts. Just some examples: Saccharomyces krusei, Issatchenkia orientalis
Growth in malt extract:	Cell morphology:	Ovoid to elongate form, 1.3-6 µm x 3.3-14 µm
	Clustering:	Occurring as single cells or in pairs
	Pseudohyphae:	Moderate formation
	Pellicle formation:	Heavy, dry climbing pellicles are formed
Growth in malt extract:	Colony morphology:	After 3 days: Butyrous and light-cream color
Fermentation:	Glucose:	Positive
	Galactose:	Negative
	Sucrose:	Negative
	Maltose:	Negative
	Lactose:	Negative
	Raffinose:	Negative
	Trehalose:	Negative

That’s all about Pichia kudriavzevii so far. In summary, Pichia kudriavzevii seems to be involved in various beverage and food fermentations and is of clinical importance. Although the clinical importance mainly affects people with a suppressed immune system. It is not clear at this point if only a certain type of strain(s) (mainly the ones isolated from animals or humans) are potentially pathogenic or all the Pichia kudriavzevii strains.

Due to the potential pathogenic character as well as the limited range of sugars Pichia kudriavzevii can utilize, I don’t see any reasons to intentionally try this yeast for beer production. Furthermore, I do not support the idea to try the yeast for any beverages. Even if you could make beer out of wood…

Bibliography

• Chan GF, Gan HM, Ling HL, Rashid NA. (2012) Genome sequence of Pichia kudriavzevii M12, a potential producer of bioethanol and phytase. Eukaryot Cell, 11(10)
• Daniel HM, Vrancken G, Takrama JF, Camu N, De Vos P, De Vuyst L (2009) Yeast diversity of Ghanaian cocoa bean heap fermentations,FEMS Yeast Research, 9, 774–78
• De Vuyst L, Neysens P (2005) The sourdough microflora: biodiversity and metabolic interactions. Trends in Food Science & Technology, 16, 43–56
• Kurtzman CP, Fell JW, Boekhout T (2011) The Yeasts, a Taxonomic Study. Volume 1. Fifth edition. Elsevier (Link to sciencedirect)
• Meroth CB, Hammes WP, and Hertel C (2003) Identification and Population Dynamics of Yeasts in Sourdough Fermentation Processes by PCR-Denaturing Gradient Gel Electrophoresis. Applied and Environmental Microbiology, 69(12)
• N’guessan KF, Brou, K, Jacques N, Casaregola S, Dje, KM (2011) Identification of yeasts during alcoholic fermentation of tchapalo, a traditional sorghum beer from Côte d’Ivoire, Antonie van Leeuwenhoek, Vol99(4), 855-864

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.

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.

Fig 2: Wyeast 3068 Weihenstephan

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