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

Insight into the genome of Saccharomyces carlsbergensis

Eureka, another yeast genome got recently published (May 2014) by scientists at the Carlsberg Laboratory in Denmark: Saccharomyces carlsbergensis, the world’s first pure lager yeast used in production since 1883. I would like to review the published article and point out some interesting results. Below the full reference of the paper I am talking about.

Walther A, Hesselbart A, Wendland J (2014) Genome Sequence of Saccharomyces carlsbergensis, the World’s First Pure Culture Lager Yeast, G3, 4:783-793; doi:10.1534/g3.113.010090, http://g3journal.org/content/4/5/783.full

The scientists sequenced the genome using next generation sequencing techniques and compared the genome with Saccharomyces cerevisiae (top-fermenting yeast) and Saccharomyces eubayanus (likely to be a parent of the lager yeasts). Lager yeasts are hybrids and resulted from an interbreeding event between a top fermenting S. cerevisiae yeasts parent as well as a non-cerevisiae parent (likely to be S. eubayanus). This means, the genome of lager yeasts consist of parts of the S. cerevisiae genome as well as parts of a non-cerevisiae parental genome.

Beside S. carlsbergensis, the authors re-sequenced another lager yeast (Weihenstephan WS34/70) for comparative reasons. Lager yeasts can be grouped into group I (Saaz-type lager yeasts) and group II (Frohberg-type lager yeasts). Members of the two groups reflect geographic associations with breweries like group I (Czech and Carlsberg) and group II (Weihenstephan and Heineken). The sequenced S. carlsbergensis strain (CBS1513) belongs to group I whereas WS34/70 belongs to group II. Comparing the two genomes therefore might give some insight into genetical differences between the two lager yeast groups.

Loss of parental S. cerevisiae DNA in S. carlsbergensis

The authors found substantial genome size differences between the two lager yeasts (about 3.5 Mb). A previous investigation showed, the Weihenstephan lager yeast contains two complete parental genomes (S. cerevisiae and S. eubayanus) with some losses at chromosome ends [Nakao et al, 2009]. To address how much of the S. carlsbergensis genome is from S. cerevisiae and S. eubayanus, the authors mapped the obtained S. carlsbergensis genome to the two parent yeast genomes. The comparison revealed, the genome of S. carlsbergensis does not contain information of the S. cerevisiae chromosomes VI, XI and XII (Fig 1, left), harbours some translocated S. eubayanus chromosomes (II, IV and VIII and XV) (Fig 1, right) as well as loss of heterozygosity in some S. cerevisiae chromosomes (Fig 1, right, chromosomes IV, XIII, XV and XVI). This lack of chromosomal information of S. cerevisiae chromosomes VI, XI and XII as well as some loss of heterozygosity is sufficient to explain the smaller genome size of S. carlsbergensis in comparison with the Weihenstephan lager yeast.

F3.large

Fig 1: Pairwise comparison of S. carlsbergensis genome with sub-genome of S. cerevisiae (left) and S. eubayanus (non-cerevisiae) sub-genome (right). Taken from Walther et al, 2014

Summarized, S. carlsbergensis (group I) lost some S. cerevisiae DNA which is still present in the Weihenstephan lager yeast (group II).

Chromosomal map of S. carlsbergensis

The authors generated a chromosomal map for S. carlsbergensis strain CBS 1513 which consists of 29 different chromosomes (Fig 2). Whereas the Weihenstephan lager yeasts harbours 36 different chromosomes (not shown). The individual chromosomes either contain only chromosomal information from the parental S. cerevisiae (parts in blue) or S. eubayanus (orange parts) yeasts or contain information from both yeasts (translocated chromosomes).

F7.large

Fig 2: Chromosomal map of S. carlsbergensis strain CBS 1513. Blue parts represent S. cerevisiae sub-genome, orange parts the S. eubayanus sub-genome. Taken from Walther et al, 2014

To investigate if group I lager yeasts resulted from a hybridization event of two haploid (one copy of each chromosome) S. cerevisiae and S. eubayanus cells, the authors determined the copy numbers of each chromosome present in the S. carlsbergensis genome. If this would be the case, one would expect to find a 1:1 ratio of S. cerevisiae and S. eubayanus chromosomes in the S. carlsbergensis genome.

Surprisingly, the S. carlsbergensis genome seems to be triploid (three copies) with one copy of S. cerevisiae and two copies of S. eubayanus genome (1:2 ratio). The complete S. carlsbergensis genome therefore consists of a total of 47 chromosomes (Fig 2). In comparison, the Weihenstephan lager yeasts is tetraploid (4 copies) with two S. cerevisiae and two S. eubayanus genomes (1:1 ratio).

The comparison showed a clear distinction between lager yeast group I and II with loss of S. cerevisiae DNA in group I. In terms of origin, one may suggest that group I lager yeasts were generated by a fusion event of a haploid S. cerevisiae with a diploid S. eubayanus yeast cell whereas group II lager yeasts originated from a diploid-diploid fusion generating tetraploid group II lager yeasts. Three conserved translocation events in both sequenced lager yeasts may however suggest a common ancestor of both lager yeast groups. And a DNA elimination event may have created group I lager yeasts afterwards.

There you have it. A pretty cool research project. I would like to finish with yet another astonishing result. The authors addressed the level of diversity of possibly one of the original S. carlsbergensis yeast strain isolated by Emil Chr. Hansen in the late 19th century (obtained from Carlsberg bottles of the late 19th century) with the strain deposited at CBS in 1947. The yeasts present in the bottles were identical with the CBS deposited yeast strain. “This suggests very limited evolution of pure cultured yeast strains under industrial fermentation conditions” [cited from Walther et al, 2014]. Pretty cool, right?

I hope you enjoyed reading my short review. Please have a look at the original genome paper as well. I think it is very well written publication. Hope to see some new lager yeast genomes coming out soon.

Bibliography

Nakao Y, Kanamori T, Itoh T, Kodama Y, Rainieri S et al (2009) Genome sequence of the lager brewing yeast, an interspecies hybrid. DNA Res. 16:115-129

BBA/EBY Brett Experiment Update 5

Eureka, we are back to Brett experiment updates. I would like to take the opportunity here to remind people to fill in evaluation data they have so far. At this point, only 6 people (out of 38 collaborators) sent in data either via the provided google doc form or by posting their results on blogs. If you haven’t sent in any data so far, may I kindly ask you to do so? Sending me your evaluation sheets as PDF works as well.

That’s it already. More yeast posts to come soon.

Hello, my name is Debaryomyces hansenii

Eureka, we are back to spoilage yeasts. Today, I would like to introduce another spoilage yeast called Debaryomyces hansenii (anamorph Candida famata). This yeast was originally isolated from saline environments and is maybe one of the most osmotolerant (can tolerate high levels of salt and sugar) yeasts in existence [Kumar, 2012]. This yeast is very common in various food products and has a big biotechnological potential. It is therefore of no surprise that two strains of this yeast, CBS767 and MTCC 234, have been previously sequenced and their genomes are published [Lépingle, 2000; Kumar, 2012].

Older publications talk of two yeast varieties of D. hansenii: D. hansenii var. hansenii and D. hansenii var. fabryi with differences in their 26S rRNA gene as well as their temperature preferences [Breuer, 2006]. The current nomenclature reinstated D. fabryi as a separate family based on the genetical differences between the two varieties. I will therefore only discuss D. hansenii: D. hansenii var. hansenii in this post.

Where do I work?

D. hansenii is the most prevalent yeast in dairy and meat products as well as early stages of soy sauce fermentation [Kurtzman, 2011]. Various isolates exist originating from cheese, sake moto, edomiso, rennet, psoriasis, infected hands and salmon [Kurtzman, 2011]. In general, D. hansenii can be found in habitats with low water activity as well as in products with high sugar concentrations [Breuer, 2006]. Although D. hansenii is considered a non-pathogenic yeast, various clinical cases of D. hansenii exist.

What about beer?

I could not find a source discussing the use of D. hansenii in the production of beer.

What is so special about me?

As already mentioned, D. hansenii can tolerate very high levels of salt. Some sources cite salinity levels up to 24% whereas Saccharomyces cerevisiae commonly tolerate levels up to 10% [Lépingle, 2000]. Such high tolerances are not that common in living organisms and can be used on industrial scale by cultivating D. hansenii at high salt levels to prevent the growth of other yeasts (quasi non-sterile production conditions). Beside dealing with high osmolarities, D. hansenii secrete toxins capable of killing other yeasts [Breuer, 2006].

Although this yeast is already an extremophile in terms of osmolarity, it does not stop there. Besides the normal sugars, D. hansenii is capable of metabolizing n-alkanes, melibiose, raffinose, soluble starch, inositol, xylose, lactic acid and citric acid [Breuer, 2006; Lépingle, 2000; Kumar, 2012]. Furthermore, this yeast can form arabitol as well as riboflavin (vitamin B2) [Génolevures, 2014]. D. hansenii is therefore used on industrial scale to produce vitamin B2 and has a big potential for other biotechnological processes.

D. hansenii is a very common yeast in cheeses and seems to have a major impact on the development of the microflora as well as the taste [Lépingle, 2000]. As previously mentioned, D. hansenii can metabolize lactic acid, citric acid and galactose. The assimilation of lactic acid by yeasts has been shown to have an impact on the bacterial flora of the cheese in types such as Limburger, Tilsiter, Port Salut, Trappist, Brick and the Danish Danbo [Breuer, 2006]. Furthermore, D. hansenii forms volatile compounds associated with a “cheesy” flavor [Breuer, 2006]. For example, D. hansenii seems to have a major role in the development of Cheddar and Camembert cheese by synthesizing S-methylthioacetate (most prevalent volatile sulphur compound found in cheese) [Breuer, 2006].

Summarized, D.hansenii is involved in various dairy products and has some very unique biochemical properties. This makes this yeast very interesting for biotechnological processes such as the production of toxins as therapeutic agents, produce xylitol (as already discussed in the previous post about Pichia kudriavzevii), manufacturing chemical compounds and the production of vitamin B2.

As soon as a yeast gets interesting on industrial scale, a genome sequencing project gets commonly initiated to get more insight into the organism you want to deal with. However, very often such genomes do not get published to keep the obtained information a secret. Luckily for us, the two draft genomes of D. hansenii can be accessed by anyone.

D. hansenii strain CBS767 (isolated from Sherry in Denmark) has been sequenced by the Génolevure project. The obtained assembly consists of 7 chromosomes (assembly size of 12.2 Mb). 205 tRNA genes could be found corresponding to a set of 43 tRNAs. D. hansenii uses an alternative genetic code and uses the codon CAG for serine instead of leucine and carries a single copy tRNA-Ser CAG.

D. hansenii strain MTCC 234 (isolated from New Zealand soil) has been Illumina sequenced and assembled using Velvet 1.1.06 into a draft genome consisting of 542 contigs and a N50 contig length of 68,507 bp [Kumar, 2012]. The authors furthermore predicted 5,313 proteins and could find matches (E-value cutoff of 10-6) in the nr NCBI database for >99.5% of the predicted proteins.

Where can you find me?

In theory, it should be easy to pick up D. hansenii from dairy products if one follows the protocol mentioned in the section below.

Some biochemical stats about me for yeast ranchers

Breuer et al mention a simple protocol to pick up D. hansenii: Cultivate yeasts at 10% NaCl and 5% glucose to discriminate between D. hansenii and other ascomycetous yeasts. Below a summary of the biochemical properties of D. hansenii. Data is summarized from Kurtzman et al (2011).

Systematic name: Debaryomyces hansenii (anamorph Candida famata)
Synonyms: There are a lots of accepted synonyms for this yeasts. Just some examples: Saccharomyces hansenii, Debaryomyces gruetzii, Pichia hansenii
Growth in malt extract: Cell morphology: Spherical to short-ovoid form, 2-7 µm x 2-9 µm
Clustering: Occurring as single cells, pairs or short chains
Pseudohyphae: Poor formation
Pellicle formation: Not described
Growth in malt extract: Colony morphology: After 4 weeks: Grayish-white to yellowish, glistening or dull, butyrous and smooth or wrinkled
Fermentation: Glucose: Weak
Galactose: Weak
Sucrose: Weak
Maltose: Weak
Lactose: Negative
Raffinose: Weak
Trehalose: Weak

That’s all about Debaryomyces hansenii. In summary, Debaryomyces hansenii is involved in the maturation process of various food products such as cheese, sausages, various other fermented products as well as industrial applications such as the production of vitamin B2.

Although this extremophilic yeast sounds really interesting, I would not use D. hansenii as a single yeast species to ferment a beer. Simply because it is a very weak fermenter of maltose and glucose, the main sugars present in wort. On the other hand, one can only guess its impact if used as a secondary or bottling strain. Maybe use this yeast for a Imperial Gose with a salt content of about 20%? Let me know if there is someone crazy enough to make such a big Imperial Gose similar to sea water…

Bibliography

  • Breuer U, Harms H (2006) Debaryomyces hansenii – an extremophilic yeast with biotechnological potential. Yeast, Vol 23, p.415-437
  • Génolevures, Debaryomyces hansenii entry (via http://genolevures.org/deha.html), accessed April 2014
  • Kumar S, Randhawa A, Ganesan K, Raghava SG, Mondal AK (2012) Draft Genome Sequence of Salt-Tolerant Yeast Debaryomyces hansenii var. hansenii MTCC 234. Eukaryotic Cell, Vol11(7), p.961-962, (via NCBI)
  • Kurtzman CP, Fell JW, Boekhout T (2011) The Yeasts, a Taxonomic Study. Volume 1. Fifth edition. Elsevier (Link to sciencedirect)
  • Lépingle A, Casaregola S, Neuvéglise C, Bon E, Nguyen HV, Artiguenave F, Wincker P, Gaillardin C (2000) Genomic Exploration of the Hemiascomycetous Yeasts: 14. Debaryomyces hansenii var. hansenii. FEBS Letters, Vol 487, p.82-86

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.

XylosePathway

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.

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

Happy Birthday to Me! and News

Happy New Year to all of you first. To a new year with lots of good beer and increasing the biodiversity of yeast strains! Now back to the topic. Eureka, I got a notification today:

Happy Anniversary!

You registered on WordPress.com 2 years ago!

Well Happy Birthday to Me then. I would like to take the opportunity to thank all the 78,723 readers/viewers over the last two years. The blog gets more and more views every month and cracked the 6,000 views per month in October 2013. Well, I started this blog in January 2012 to get in contact with other yeast and bacteria hunters in the world. On one hand to share some of my results and on the other hand to get yeast ranching discussions going. And only yeast ranching. However mainly on a scientific level since I initially believed that most of the yeast ranchers out there have a scientific background. Another main goal was to publish our recipes to share the experiences we made with others.

And here am I now. Two years later to reconsider some of the points mentioned earlier. I realized after the first year of blogging that there are a lot of people out there with a non-scientific background interested in yeast ranching as well as general yeast techniques such as harvesting, storing and so on. I posted some general yeast posts in 2012 which apparently remain the most visited posts on my blog even in 2013. popular_posts_2013Judging from these results, there seems to be a demand for general information to eventually get people into yeast ranching. I will therefore focus more on yeast posts in the future instead of posting homebrew recipes and tastings. But I keep the option open. Unfortunately the blog’s name has no association with yeast. But I don’t care about that.

What can you expect to read on this blog in the future ?

  • Posts concerning other yeasts which are associated with beer and other fermented beverages. No spoilers here
  • Covering the Brettanoymces specific chemistry and enzymes involved to create the love or hate character of Brett beers
  • A series of posts about Brettanomyces in collaboration with Dmitri (bkyeast)
  • Insight into the Brettanoymces draft genome/s (will take me another while though)
  • Further posts about isolating yeasts from beer. But only very special cases
  • Preliminary data about other selective media I tested to differentiate between Brettanoymces species (repeat: differentiate between species)
  • Results form the BBA/EBY Brettanoymces experiment
  • Any interesting yeast related topics I come across

Any other news?

  • Eureka Brewing Yeast releases? Not before first results are in from the experiment. And I cannot tell how long this will take as it depends on the collaborators to hand in results. And me to go through the results to make my decisions…
  • New strains of Brettanomyces from various new sources will be included into the Eureka Brewing Yeast program. Possible candidates for another Brettanomyces experiment? Who knows
  • Eureka Brewing gets a separate German page since information about yeast are not really well documented in German. Ich arbeite seit einiger Zeit an einer Seite um Informationen, die bisher nur in Englisch auf diesem Blog publiziert wurden, auf Deutsch zu übersetzen. Grundlage hier ist ein Hefekurs den ich für einen Homebrewclub gehalten habe. Da meine Grammatik mehr Zufall ist möchten sich doch jene die sich für Korrekturen zur Verfügung stellen würden bei mir per mail melden
  • Blackwell Brewery. Still going and planning various collaborations. We are looking for a space to brew in Bern or the neighbourhood of Bern, Switzerland. Anyone with ideas for a possible location for the brewery, per mail melden
    Although read the About page first so you know what you are dealing with…
  • Any preternatural (never used that word before, if I used the word in the wrong context, per mail melden.) beers going on? Of course! Various sours are in the pipeline as well as some really strong, really light, really good, really bad and everything in-between beers
  • If you are from a small artisanal brewery in Switzerland (or anywhere else) and you are interested in a collaboration, per mail melden. Although read the About page first so you know what you are dealing with…

Thanks for viewing and reading. If you haven’t learned anything new here let me remind you that you eventually learned that “per mail melden” means “please write us a really nice, well structured e-mail with “hello” and “goodbye” and leaving your name” in German. Cheers!