Hello, my name is Metschnikowia pulcherrima

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

Where do I work?

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

What about beer?

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

What is so special about me?

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

Where can you find me?

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

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

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

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

Control:

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

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

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

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

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

Metschnikowia partial ferment:

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

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

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

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

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

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

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

Some biochemical stats about me for yeast ranchers

First some micrographs:

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

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

That’s all for today. Thanks for reading.

Bibliography

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

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

Where do I work?

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

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

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

What about beer?

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

lambic_fermentation

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

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

What is so special about me?

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

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

Where can you find me?

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

Some biochemical stats about me for yeast ranchers

Kloeckera_apiculata_001_scale.tif

Fig 2: Kloeckera apiculata (sample received from SuiGeneris)

Data summarized from Kurtzman et al (2011).

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

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

Bibliography

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

Hello, my name is Kluyveromyces marxianus

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

Where do I work?

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

What about beer?

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

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

gowe_production_flow

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

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

What is so special about me?

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

Where can you find me?

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

Some biochemical stats about me for yeast ranchers

Data summarized from Kurtzman et al (2011).

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

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

Bibliography

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

Hello, my name is 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

A glimpse into yeast flocculation

Eureka, science post! This is an entire review post about yeast flocculation. Flocculation describes the ability of yeast cells to aggregate into clumps/flocs and then drop out of suspension. This happens during the end of fermentation and the yeast cells form a sediment at the bottom of the fermenter. The flocculation treat is mainly genetically derived and thereby depends on the yeast strain. Flocculation characteristics can sometimes change and lead to early flocculation to occur or to loss of flocculation. Despite the genetics, there are a lot of ways a homebrewer can influence the yeast flocculation.

In this post I would like to cover the basic principles how flocculation functions on a genetic and biochemistry level, then speak about factors influencing the flocculation and end with some words about how a homebrewer can influence the flocculation of yeasts. Lets begin with a general overview about flocculation.

Yeast flocculation profiles can be distinguished into three groups:

  • High flocculate strain (strongly sedimenting): Flocculation starts after 3-5 days (if kept at correct fermentation temperature). These strains tend to flocculate earlier during the fermentation and form a sediment at the bottom of the fermenter. Most of the English yeasts belong into this group. Such yeasts tend to lead to lower attenuation (higher terminal gravity) and sweet beers since the yeasts cells are not in suspension anymore and in contact with the sugars. In addition, a lot of fermentation byproducts stay in the beer such as diacetyl and esters for example.
  • Medium flocculate strain (powdery): Flocculation starts after 6-15 days. Typical ale strains and lager strains. Such yeasts give you a clean and balanced beer. Such yeast stay in contact with the beer and can continue to ferment and metabolize fermentation byproducts such as diacetyl.
  • Low flocculate strain (non-flocculate): Flocculation starts > 15 days. Most of the wild yeasts, Hefeweizen- and Belgian yeasts plus some lager strains belong into this category. Such yeasts tend to stay in suspension and lead to a cloudy, yeasty beer. In addition, such strains can make filtering of beer rather difficult.

Comparing the three different groups above, it is obvious that non-domesticated yeasts (named wild yeasts) are low flocculating. The flocculation character in domesticated yeast cells got improved by selective pressure. One easy way to do so it harvest the yeast from the bottom of the fermenter and therefore only harvest the highly flocculent yeasts. More about that later on.

How flocculation in yeast works

As already mentioned, the flocculation is mainly genetically driven. I would like to start with the phenotypes first and then get into the genetical setup since it might be easier to understand the different genes and what they do.

Cell biology of flocculation

One trait that influences flocculation is the charge surface charge of the yeast cell. The surface charge is mainly negatively charged but the charge depends on the strain, the phase of growth, the oxygen content in the wort, starvation of the cell and cell age. Most of these factors can be influenced by the brewer. Due to the negatively charged surface yeast cells repel each others. Such repulsions prevent yeasts from flocculating since flocculation involves yeast cells to get in contact first. Top-fermenting strains seem to have a less negatively charged cell surface than bottom-fermenting strains (Amory and Rouxhet, 1988).

The yeast cells have a cell membrane and a cell wall. The cell membrane’s function is mainly to regulate what gets in and out of the cell. The cell wall’s job is to stabilize the whole cell and is therefore responsible for the integrity of the yeast cell. One of the most important building blocks is mannan. We will come back to the cell membrane and mannan later on.

Non-flocculent yeast cells appear as smooth cells on a SEM (scanning electron microscope) micrograph and flocculent yeast cells appear to have some sort of hairs. Non-flocculent yeast strains collide but don’t form clumps. On the other hand, flocculent strains form clumps if they collide. As previously mentioned, yeast cells are in general negatively charged and therefore repel each other. What is the reason for the flocculent yeast strains to form clumps then?

Biochemistry of flocculation

Yeast cells like mammalian cells have a lot of surface proteins on/in their cell membranes. Such proteins are necessary for the yeast strains for signalling (interacting with the environment) and get molecules into and out of the cell. One could easily write books only about surface proteins and that’s why I will not get into further details here. One possible way to explain the interaction of flocculent yeasts is the lectin hypothesis.

Fig1

Fig 1: Mannose and glucose structures

This hypothesis states that controlled interactions of specific surface proteins between different yeast cells are involved in the flocculation. One such protein is called zymolectin which is produced by the yeast cell and then incorporated into the cell wall. As one can tell zymolectin belongs to the family of lectins which is a family of proteins that bind sugars. Zymolectin can bind the sugar molecule mannose (Fig 1). In addition, it can also bind to mannan, the building block of the cell wall (Fig 2), which is made from mannose molecules. A bond between zymolectin and mannan (from different yeast cells) therefore links two cells together and initiates the formation of yeast flocs.

Fig2

Fig 2: Mannan structure

The critical step for the flocculation to occur is the point where zymolectin gets active and establishes the connection to another yeast cell. Not much is yet known about the zymolectin expression. Zymolectin may become active at the end of exponential growth and might be triggered by depletion of nutrients such as sugars and an increase of fermentation byproducts such as ethanol. Lets have a closer look at the zymolectin family members.

  • Flo1 (Flocculin-1): (http://www.uniprot.org/uniprot/P32768) Synonyms are FLO2 and FLO4. This protein selectively binds to mannan residues in the cell wall and is inhibited by mannose but not glucose, maltose, sucrose of galactose. The protein is 1,537 amino acids (aa) long and has a sugar recognition site between position 197- 240. Interestingly, there are 18 repeated domains (flocculin repeats) in this protein each with a length of 45 aa plus a PA14 domain which is responsible for binding sugars (Fig 3)

    Fig3

    Fig 3: FLO1 with 18 flocculin repeats (red) and a PA14 domain (blue) (Pfam)

  • Flo5 (Flocculin-5): (http://www.uniprot.org/uniprot/P38894) 1,075 aa long. The protein consists of one P414 domain, 8 flocculin domains and 3 flocculin type 3 domains. Plus a sugar binding site
  • Flo8 (Transcriptional activator FLO8): (http://www.uniprot.org/uniprot/P40068) 799 aa long. Putative transcription factor of FLO1, FLO9 and FLO11/MUC1
  • Flo9 (Flocculin-9): (http://www.uniprot.org/uniprot/P39712) 1,322 aa long. The protein consists of one P414 domain, 13 flocculin domains and 3 flocculin type 3 domains. Plus a sugar binding site
  • Flo10 (Flocculin-10): (http://www.uniprot.org/uniprot/P36170) 1,169 aa long. The protein consists of one P414 domain
  • Flo11 (Flocculin-11): (http://www.uniprot.org/uniprot/P08640) 1,367 aa long. No conserved domains found. This protein is involved in filamentous growth (see next post)
  • Lg-Flo1 (must be present in lager yeast)
  • NewFlo: These proteins bind to mannose and glucose. Mannose, glucose, maltose and sucrose can inhibit zymolectin. There are two different proteins belonging into this group of zymolectins:Lg-Flo6p: (http://www.uniprot.org/uniprot/E9P9E1) 428 aa. Not much is known for this protein. However, there is a PA14 domain and 3 flocculin repeats presentLg-Flo10p: (http://www.uniprot.org/uniprot/E9P9E2) 492 aa. Not much is known for this protein either. However yet again, one PA14 domain and 5 flocculin domains

The longer the flocculin protein (the more flocculin repeats), the stronger the flocculation is (Vidgren et al 2011). Flo1 therefore shows a strong flocculation character. The NewFLo phenotype is very common in brewer’s yeast. Lets summarize, so far three groups of flocculation phenotype have been described:

  • Flo1 type (is inhibited by mannose only). This phenotype occurs in Lager and ale yeast strains and is associated with FLO1 gene. Flocculation occurs independently on wort sugars (not suitable for brewing)
  • NewFlo type (is inhibited by mannose, glucose, maltose, sucrose). Suited for brewing. Flocculation occurs if wort sugars are metabolized.
  • Mannose insensitive. This phenotype occurs in ale but not in lager strains. Calcium ions are necessary. As it can be concluded from the name, this phenotype is not inhibited by mannose. Flocculation can be induced by low ethanol concentrations (Dengis et al, 1995). One possible mechanism for this phenotype might be by simply changing the cell surface charge. However, the evidence that small amounts of calcium are necessary and that FLO11 is involved points to an adhesion-mediated mechanism as well but not based on flocculin repeats.

In addition to the three groups, co-flocculation can occur as well if a non-flocculent and a flocculent strain get in contact. In this case the zymolectin from the flocculent strain binds to the mannose of the non-flocculent strain and pulls the non-flocculent strain down. Co-flocculation can occur with bacteria such as Acetobacter, Lactobacillus and Pediococcus as well (Vidgren et al 2011).

Genetical setup of flocculation

One to three genes are present in yeast strains which are inherited dominant. Flocculation therefore can be improved by crossing yeast strains: Cross a high flocculent strain with a low flocculent strain leads to a high flocculent yeast. Although the flocculent trait is dominantly inherited, flocculation can also decrease.

  • FLO1 (Flocculin-1) Located on chr01 and encodes Flo1 protein https://www.ncbi.nlm.nih.gov/nuccore/NM_001178230.1 4,614 bp. No introns. This gene seems to be Saccharomyces specific since I could not find any other organism with similar genes.FLO2 and FLO4 are alleles of FLO1 and FLO5, FLO9 is a homologue of FLO1. Any expression of FLO1, FLO2, FLO4, FLO5 or FLO9 leads to the initiation of flocculation of the Flo1 phenotype.
  • Lg-FLO1 can be found in Lager yeasts and is responsible for the NewFlo phenotype

The FLO genes are relatively unstable due to mutations and the highly repetitive pattern due to flocculin repeats. Highly repetitive sequences in the genome change more rapidly than regions with less repetitive motifs (Vidgren et al, 2011). A lot of mutations happen in the FLO genes and the most commons ones lead to deletions or any other alterations leading to a decrease of flocculation. In addition, FLO genes are near telomeres (ends of chromosomes) and can get transcriptionally silenced. Nevertheless, flocculation not solely relies on the FLO genes but implies physical interactions of yeast cells (collision of yeast cells).

Putting it all together. For flocculation to occur the following factors have to be true:

  • Flocculins have to be expressed by the yeast and present in the cell wall (for Flo1 and NewFlo type)
  • Physical interaction between yeast cells
  • Absence of inhibitory sugars (in NewFlo type)
  • Small amounts of calcium ions present. Calcium is necessary for the correct conformational shape of the zymolectin molecules
  • Right environmental conditions

Environmental factors influencing flocculation

Now that we covered the biochemistry and genetics lecture part about flocculation, let’s have a look at some environmental factors affecting flocculation.

What environmental factors influence yeast flocculation?

  • Fermentation temperature
    • Lower temperatures seem to initiate flocculation as well as higher temperatures above the recommended fermentation temperatures
  • Wort pH. Top-fermenting yeast strains flocculate within a pH range of pH 3 – 4.5, bottom fermenting ones between pH 3.5 – 6
  • Original gravity
  • Oxygen content added
    • Poor wort aeration can result in an early flocculation. Oxygen content at pitching increases sterol and fatty acid content in cell membrane and increases the cell surface hydrophobicity
  • Depletion of inhibitory sugars such as sucrose, glucose, maltose (all inhibit flocculation in NewFlo type only)
  • Increase of fermentation byproducts such as ethanol can influence flocculation as well
  • Factors increasing the chance that yeast cells collide
    • Pitching rate (higher pitching rate gives a higher yeast cell density)
    • turbulence by carbon dioxide production
    • Yeast age. Older yeast cells tend to have a rougher cell surface due to the undergone budding events and are therefore prone to stick to other cells
  • Factors decreasing the cell surface charge (decrease of electrostatic repulsion)
    • Ethanol concentration
    • pH of wort
    • Changes in cell wall composition
    • Expression and incorporation of flocculins into the yeast cell wall
  • Premature yeast flocculation-inducing factors (PYE) from the barley husks can lead to premature flocculation. Barley produces PYE as a response to microbial growth during the steeping process. Further investigations are necessary to fully understand the PYE influence on flocculation

This list might look very frightening to homebrewers. A closer look reveals some common factors which can be broken down into:

  • Adequate oxygenation of the wort. Poor oxygenation not only leads to possible off-flavors but to incomplete fermentation due to delayed flocculation and reduced sterol content in the cell membranes
  • Temperature. Flocculation is temperature dependent. In general a lower temperature favors yeast flocculation. However, this is very yeast strain dependent
  • Pitching rates. Higher pitching rates increase amount of older cells and therefore favors flocculation. However, I do not recommend to overpitch to improve the flocculation character of a yeast strain

Beside oxygenation, temperature and pitching rates, how can a homebrewer lower the changes to encounter problems due to different flocculation behaviour?

  • Choose the right yeast strain. If you plan on brewing a clear beer, better stick to a yeast strain with a high to very high flocculation potential. Flocculation behaviours can be looked up on the yeast suppliers webpages
  • Decrease temperature to 0°C (32°F) after the fermentation reached terminal gravity. Lowering the temperature results in higher flocculation rates and leads to clearer beers. Don’t chill the beer too early
  • Get yeast out of beer by filtration or centrifugation (if you can’t wait for the yeast to drop out itself)
  • Add collagens (positively charged) and pull down the yeast cells. This is commonly used in real ales by adding Isinglass. By doing this, one can use the positive character a low flocculate yeast strain might contribute to a beer without having a cloudy pint of beer in the end
  • Collect yeast from the bottom of the fermenter or from kräusen and thereby select for the highly flocculate yeast cells. If you collect yeast from the yeast cake, the most flocculate yeasts will be in the middle part of the yeast sediment. The non-flocculate or poorly flocculate yeasts will be in the top layer and older cells, dead cells in the bottom layer
  • Yeast storage. Use a method without excessive stressing the yeast cells such as low/high osmolarity of the storage media. Storing yeast at lower temperatures (4°C) can result in reduced flocculation. However, these effects are strain dependent
  • Keep acid washing steps at a minimum. Washing cells with acid can change the surface protein composition and therefore might have an impact on the surface charge and surface hydrophobicity
  • Avoid excessive re-pitching of the same yeast over and over again. Don’t re-pitch your yeast for more than 5 – 10 times.

What to do if your yeast does not flocculate as before?

  • A change of flocculation behaviour can have several causes such as mutations, mixed cultures (infections), different environmental factors. Finding the cause for the different flocculation behaviour might be hard. Therefore:
  • Don’t use the same strain for another batch of beer. Start with a fresh yeast
  • If a high flocculent strain is used, get the yeast back into suspension by either swirling or venting some carbon dioxide into the fermenter

To keep in mind:

  • Flocculation character of a yeast directly impacts the flavor and fermentation performance of a beer. Therefore choosing the right flocculate yeast strain is very important in the first place
  • Keep as much of the fermentation factors as consistent as possible. This includes fermentation temperatures, pitching rates, oxygenation etc.
  • Keep record to be able to observe changes in flocculation
  • Flocculation itself depends on yeast strain and its FLO genes, environmental factors and the physical interaction between yeast cells

Flocculation seems to be Saccharomyces yeast specific and a lot of research is still done to further understand how flocculation works. I hope I could give you a small glimpse into the topic and got you an idea what flocculation is all about. Including some advice what influences flocculation and what a (home)brewer can do about it to keep flocculation behaviours as constant as possible. The next post concerning flocculation will cover the biological function of FLO genes and therefore the biological function of flocculation for the yeasts cells and further insights into other flocculins and their biological role in Saccharomyces. Cheers!

References:

  • Amory DE, Rouxhet PG (1998) Surface properties of Saccharomyces cerevisiae and Saccharomyces carlsbergensis: chemical composition, electrostatic charge and hydrophobicity. Biochim. Biophys. Acta, 938: 61 – 70
  • Dengis PB, Nélissen LR, Rouxhet PG (1995) Mechanism of Yeast Flocculation: Comparison of Top- and Bottom-Fermenting Strains. Appl Environ Microbiol, 61(2): 718 – 728
  • Fix G (1999) Principles of brewing science: a study of serious brewing issues. Brewers Publication, 2nd edition
  • Narziss L (2005) Abriss der Bierbrauerei. WILEY-VCH, Weinheim, 7th edition
  • Verstrepen K, Industrial Microbiology Part II – Fermentation, Katholieke Universiteit Leuven, http://www.biw.kuleuven.be/dtp/cmpg/G%26G1/assets/internal/IM-class2-Fermentation-v4.pdf
  • Verstrepen KJ, Derdelinckx G, Verachtert H, Delvaux FR (2003) Yeast flocculation: what brewers should know. Appl. Microbiol. Biotechnol, 61: 197 – 205
  • Vidgren V, Londesborough J (2011) 125th Anniversary Review: Yeast FLocculation and Sedimentation in Brewing. J. Inst. Brew. 117(4): 475 -487
  • White C (2012) Flocculation Basics. www.whitelabs.com/beer/Flocculation_help.pdf
  • White C, Zainasheff J (2010) Yeast: The Practical Guide to Beer Fermentation. Brewers Publication, 1st edition
  • Wikipedia (2012) Flocculation, http://en.wikipedia.org/wiki/Flocculation
  • Wyeast (2012) Flocculation/Clarification http://www.wyeastlab.com/hb_clarification.cfm