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?

As 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)).

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:

Yeast 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

• Kurtzman CP, Fell JW, Boekhout T (2011) The Yeasts, a Taxonomic Study. Volume 1. Fifth edition. Elsevier (Link to sciencedirect)
• Oro L, Ciani M, Comitini F (2014) Antimicrobial activity of Metschnikowia pulcherrima on wine yeasts. J Appl Microbiol. 116(5):1209-17
• wineserver.ucdavis.edu (2015) http://wineserver.ucdavis.edu/industry/enology/winemicro/wineyeast/metschnikowia_pulcherrima.html

Brettanomyces Phylogenetic II & WLP Brett Trois crisis

Starting 2015 with science. This is a more in depth post about yeast phylogeny. Please have a look at the more basic post here. This time, I would like to discuss the relationship of various Brettanomyces/Dekkera strains and share my results concerning the recent WLP Brettanomyces bruxellensis Trois yeast ID crisis.

I would like to start with the Brettanomyces/Dekkera strains first. I obtained 38 Dekkera/Brettanomyces sequences (26S rDNA) from the CBS database, aligned them using MUSCLE (run in default mode, see MSA in Fig 1) and reconstructed a phylogenetic tree using MABL (model HKY85) and rendered using TreeDyn (run in default mode, Fig 2).

Fig 1: MUSCLE multiple sequence alignment (MSA) of 26S rDNA sequences from 38 different Brettanomyces/Dekkera strains obtained from CBS

I would like to emphasize the importance of having a look at the intermediate steps in constructing a phylogenetic tree since sequence alignments can happen in various ways. Looking at the MUSCLE output, one can already expect to see the individual strains clustered together due to their sequence similarities. However, there is one particular sequence that seems to be a bit off (sequence number 1, B. naardenensis CBS 6116). This sequence seems to be different from all the other B. naardenensis sequences shown at the bottom (sequences 29-38). Based on this result, one can expect to see CBS 6116 to be an out-group to the other B. naardenensis sequences. In summary, the alignments seem to be okay and let’s have a look at the phylogenetic tree of the 38 sequences.

Fig 2: Brettanomyces/Dekkera phylogenetic tree using 26S rDNA sequences obtained from CBS. Branch length is proportional to the number of substitutions per site (values shown in red)

As already observed in the MSA, the individual Brettanomyces/Dekkera species cluster together as expected (Fig 2). B. naardenensis CBS6116 does indeed form an out-group (isolated from lemon drink in France) and is kind of distant to the other B. naardenesis sequences. So why is this CBS 6116 sequence different?

To address this question, I tried to figure out first what other sequences are similar to CBS 6116 by BLASTing against the NCBI nr database (run in default mode). I got several hits and manually inspected the results via alignment. The CBS 6116 sequence has high sequence identities to Pichia guillermondii (Fig 3). One might reason – based on these results – that CBS 6116 is not a Dekkera/Brettanomyces strain.

Fig 3: MSA B. naardenensis sequences & Pichia guilliermondii

Going back to the phylogenetic tree in Fig 2. It seems that all the current Dekkera/Brettanomyces species end up in the same clusters and show enough substitutions to tell individual species apart. This is very important information for everyone interested in determining the species of an unknown Dekkera/Brettanomyces strain.

WLP Brettanomyces Trois ID crisis

I got interested in the WLP story (WLP Brettanomyces bruxellensis Trois not being a Brettanoymces strain) and started by looking at the WLP Brett Trois ITS sequences I received from Omega Labs (reads 64-ITS1.ab1 and 64-ITS4.ab1). I had a quick look at the chromatograms and would not use any of the reads for my own work due to its low base quality and multiple peak calls at certain positions. Besides the read from Omega Labs, I received a read used to ID the B. Trois as Saccharoymces cerevisiae at Charles River’s Lab as well as the read published by SuiGeneris. Since I don’t have the chromatograms for these two sequences, I manually inspected the reads in various alignments to get an idea about their quality. Lets start with my analysis for the WLP Brettanoymces bruxellensis Trois ID’ing based on phylogenetic trees.

To get the most likely phylogenetic tree one has to follow some basic rules. Beginning with looking at the same shared derived homologous traits (homologous DNA sequences) and verifying that no other DNA sequence alterations impact the phylogenetic tree like sequencing errors (wrong base calls, no base call, multiple base calls etc). So far so good. I aligned some ITS2 regions from various CBS Saccharomyces strains to the various ITS2 reads from WLP’s Brettanomyces Trois (Fig 4).

Fig 4: MSA of ITS2 rDNA sequences from various Saccharomyces sp. strains obtained from CBS and ITS2 sequences from WLP B. Trois

Lets have a look at the alignment shown in Fig 4. Especially at the 64-ITS4.ab1 read from WLP B. Trois (sequence 1 shown at the top). One can easily see, that various base differences exists compared to the other sequences in the alignment. Supporting the initial idea that the DNA sequence from this read is not very reliable nor very representative (if one compares the read to the two other WLP B. Trois reads shown at the bottom). Due to the differences in this read, I would not be surprised to see this read out-grouped in the phylogeny tree later on. The two reads from Charles River’s Lab and SuiGeneris seem to be way better and similar to other Saccharomyces sp. sequences shown in the alignment.

To make the phylogeny more computational efficient and more reliable, I extracted the sequences mapping to the two WLP sequences (sequence regions from the right side in Fig 4) to receive the alignment shown in the next figure (Fig 5).

Fig 5: MSA of ITS2 rDNA sequences from various Saccharomyces sp. strains obtained from CBS (extracted)

A first look at the alignment in Fig 5 reveals some hotspots for variations like gaps and different base calls (color regions). The question I would like to address now is whether the variations are due to speciation or artifacts. Artifacts are commonly more random than variations due to speciation. A quick inspection reveals lots of random variations in the 64-ITS4 read but none/few for the two other WLP B. Trois reads.

The phylogeny tree obtained for the sequences shown in the alignment in Fig 5 is shown in Fig 6

Fig 6: Saccharomyces phylogenetic tree using ITS2 sequences obtained from CBS & WLP B. Trois from CharlesRiverLab/SuiGeneris and n64-ITS4.ab1 from Omega Lab. Branch length is proportional to the number of substitutions per site (values shown in red)

As expected the n64-ITS4 read gets out-grouped and might be interpreted as a different species. Well this is true if one just looks at the tree in Fig 6 but did not look at the previous alignments and possible reasons for the out-grouping. In this case, the quality of the base calls for the n64-ITS4.ab1 read are very poor and led to wrong base calls after all (investigated by pair-pair alignments). In summary, the differences leading to an out-grouping of WLP B. Trois based on the n64-ITS2 read are not due to the physical differences in the DNA sequence but due to sequencing errors.

In summary, the two WLP B. Trois sequences group together with other Saccharomyces cerevisiae strains. Supporting the current view that WLP B. Trois might indeed not be a Dekkera/Brettanomyces strain (at least based on ITS2 sequence homology). Or at least the samples of WLP B. Trois that float around these days.

As I do isolate and work with my own Brettanomyces strains, it happened to me various times that I was able to observe some Saccharomyces cerevisiae beside the initial Brettanomyces strain after some serial re-pitches (and I did not use Saccharomyces for the fermentation). This Saccharomyces contamination might lead to problems during the propagation. Saccharomyces cerevisiae might overgrow Brettanomyces increasing the Saccharomyces:Brettanomyces ratio even further. Eventually leading to very few Brettanomyces cells left in a population. Keeping Brettanomyces samples is not as easy as one might think. And I would not be surprised if more yeast conundrums turn up in the next years.

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].

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

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.

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.

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).

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

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