# Tasting: Mikkeller’s Yeast Series 2.0

Eureka, I would like to share some of my tasting experiences of Mikkeller’s Yeast Series 2.0. The basic idea behind this series was to compare different yeast strains and their effects on the beer’s aroma and taste. I could get my hands on five of the six beers in the series (English Ale yeast is missing) and did a side-by-side tasting.

The base beer was all the same. In one case, the beer was fermented with a Lager strain, another one with an American Ale strain, yet another one with a Saison strain and two with Brettanomyces lambicus and Brettanomyces bruxellensis respectively. Lets see how they tasted and the individual strain’s impact on the flavor profile.

Lager yeast

Aroma: Very hoppy aroma (lots of grapes, fruits). The combination of all the hops used (Simcoe, Nugget, Warrior, Amarillo and Centennial) remind me of Nelson Sauvin hops. No yeast character.

Appearance: Orange, clear, 1 finger white head, nice bubbling.

Flavor: Fruity, nice bitterness level.

Mouthfeel: Medium body, average carbonation level, bitter/fruity aftertaste and a grassy finish

Overall Impression: Rather clean beer (in terms of yeast character). Very pronounced hop aroma and bitterness and a grassy finish

American Ale yeast

Aroma: Less hoppy than Lager example. Even a musty component in there. Doesn’t smell clean at all.

Appearance: Orange, clear, 1 finger white head, nice bubbling.

Flavor: Luckily nothing of the weird musty aroma is on the palate. Very fruity beer with a well-balanced bitterness. No typical yeast character.

Mouthfeel: Medium body, average carbonation level, bitter/fruity aftertaste. No grassy finish

Overall Impression: Compared to the Lager version, this beer is smoother in terms of bitterness. The bitterness is well incorporated and there is no grassy finish. However, the aroma in this beer is not as nice. We could not detect any yeast character in this example.

Saison yeast

Aroma: Pine, lots of tropical fruits and citrus and some spicy character (pepper).

Appearance: Orange, clear, 1 finger white head, nice bubbling.

Flavor: Again some fruits and some spiciness in addition.

Mouthfeel: Medium body, average carbonation level, slight bitter aftertaste and a grassy finish and even a bit astringent.

Overall Impression: Slightly different aroma compared to the previous two examples. This time, we could detect some yeast specific character (pepper). This yeast seems to accentuate the bitterness in the aftertaste including a grassy, astringent finish.

Brettanomyces lambicus yeast

Aroma: Subtle hop aroma, no funk…

Appearance: Orange, clear, 1 finger white head, nice bubbling.

Flavor: A bit of a disappointment. Subtile fruity beer with a well-balanced bitterness. No typical yeast character and no funk. Actually a rather clean beer.

Mouthfeel: Medium to low body, average carbonation level, slight bitter aftertaste.

Overall Impression: Not very funky nor very interesting. Average beer. We could not detect any yeast character.

Brettanomyces bruxellensis yeast

Aroma: Wow, now we are talking. There is some Brett funk going on: Wood notes, horse blanket, slight vinegar and the hop profile in the back. This beer reminds me of Cantillon’s Iris with Nelson Sauvin hops instead of the Saaz hops they use. Simply amazing smell!

Appearance: Orange, clear, 1 finger white head, nice bubbling.

Flavor: Unfortunately, not a lot of funk on the palate. Some leathery notes are present. Some fruity notes as well and a well incorporated bitterness. Rather clean beer.

Mouthfeel: Medium to low body, average carbonation level, no bitter nor grassy aftertaste. Hint of tartness reminds of the Brettanomyces in this beer.

Overall Impression: Judging from the smell, the most interesting one in the series for sure. B. bruxellensis really shows itself here. The aroma profile of this beer is surprisingly complex in my opinion. The flavor on the other side is not very yeast pronounced. But the finish is rather pleasant again.

What we learned from this tasting:

Lager strain: Gives a hop forward beer. Clean and very pronounced hop aroma. More pronounced bitterness and a grassy finish.

American Ale strain: Well incorporated bitterness and nice finish. This strain seems to work for more hop forward beers.

Saison strain: Some yeast specific character in the nose and palate. This strain accentuates the bitterness and leads to a grassy and astringent finish. Not really working for me. The spicy character, the grassy thing and the astringency makes it hard to enjoy this beer.

B. lambicus strain: Not a very funky Brett strain. Rather clean beer (compared to B. bruxellensis version). A side note. This doesn’t have to be true for every B. lambicus strain. There are so many B. lambicus strains with different flavor profiles.

B. bruxellensis strain: Lots of Brett character in the nose. But not so much on the palate. Rather clean and smooth beer with a nice bitterness level and no grassy finish.

I will put some efforts into brewing something like the B. bruxellensis beer myself. I am really fascinated about the complexity one can get with a single Brettanomyces fermented beer. Unfortunately, I tried to isolate some yeast from different Mikkeller beers before (brewed by DeProef) but never managed to recover any viable yeasts from the sediments in the bottles. I guess all the DeProef’s beers are pasteurized and therefore no (or a very small) chance to get any living yeasts out of bottles. That’s why I did not bother to isolate the B. bruxellensis strain at all. Thanks for reading, commenting and stay tuned!

# 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.

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.

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)

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
• 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

# About the morphology of colonies

Eureka, today’s post covers some general information about the morphology of bacteria, yeasts and other microorganism on agar plates and why it is important to know at least a bit about it to get the most information out of your agar platings.

Q: What do you mean by morphology of colonies?

The morphology of a colony describes how microorganisms appear on agar media such as Sabouraud, malt agar etc. Morphology just describes the colonies. If you streak some microorganisms on agar plates, they grown (if the media is appropriate for this particular organism) and form visible colonies. The colonies appear as spots like shown in Fig 1. It is important to remember that a colony are thousands to millions of microorganisms together, not a single microorganism cell. Ideally all the cells within a colony originated from one single cell at the beginning (clonal expansion). If single cells are closer together on the agar, the individual colonies overlap and no single colonies are visible (left-upper part in Fig 1). In this case, the concentration of the yeasts is just too high to observe individual colonies.

Fig 1: Brettanomyces bruxellensis on Sabouraud agar plate after 11 days

Fig 1 shows what you get if you streak some Brettanomyces yeast on Sabouraud agar. The roundish spots are the colonies (as you can see on the right side in Fig 1).

Q: How do you get single colonies?

To get an accurate description of a colony, single colonies are necessary. But how do you get single colonies in the first place? As mentioned above, if the individual cells after streaking are to close to each other, the colonies might overlap. To get single colonies one simply has to ensure a low concentration to prevent such colony-overlays. One example to do so is to dilute the cells directly on the plate itself by using a special streak technique called dilution streak or Z-streak (Fig 2). How this is done is shown in a video (YouTube) as well.

Fig 2: Dilution streak done with three streaks

Begin with a cell suspension. You might even use a yeast slurry in the first place. A first streak is done to get some cells on the plate (Fig 2, streak 1). One expects a lot of cells visible on the trajectory of the first streak and the individual colonies overlay each others. After the first streak, you sterilize your inoculation loop, let it cool down and collect some cells by passing the inoculation loop through the first streak for a second one. This time the concentration of cells is already lower because you only pick a subset of yeast cells. This process can be done for a second time to get three streaks in the end (Fig 2). The plate after a dilution streak might look like shown in Fig 1. Unfortunately, there are no colonies visible in the third streak anymore. Anyway, I hope you get the idea.

Single colonies are not only useful to describe their morphology but also to differentiate between different microorganisms. For instance, if you are interested in separating the Saccharomyces yeasts (brewer’s yeast) from Brettanomyces yeasts you can use the dilution streak and hopefully some colonies arise from single Saccharomyces colonies and others from Brettanomyces cells.

Q: Why is the morphology important?

Lets assume the morphology of a colony, representing one kind of microorganism (remember the concept of the single cell at the beginning), is unique for every microorganism there exists. The morphological description could therefore be used to identify the kind of microorganism on your agar plate. This is just an assumption because there are a lot of microorganisms which have similar morphologies. To summarize, the morphology of the colonies can be useful to identify the kind of microorganism you have on your plate. Lets go through some examples. Have a look at Fig 3.

Fig 3: Girardin bugs on Sabouraud agar plate

I assume it is obvious that there are different kinds of colonies and hence morphologies. There are differences in shapes, size and colors. To conclude, different morphologies originate from different microorganisms. I can give you even further information here. The white colonies (big ones and wavy) are yeast cells, the flat beige ones bacteria. The very small white colonies are another kind of bacteria. You see, the morphology can even be used to differ between yeasts and bacteria. That’s why agar media are very common in microbiology labs to identify different kinds of yeast/bacteria. One application here could be to test a beer for spoilage organisms such as Lactobacillus (beer turned sour). Plate some of the sour beer on a plate where Lactobacillus can grow and if colonies arise with a typical Lactobacillus morphology, you can be certain to have a Lactobacillus contamination in your beer. I will not get into further detail about the different media and strategies used to do these tricks. Just to give you an idea what the whole agar media method is capable of.

Fig 4: Water kefir on Sabouraud agar plate

Maybe an example to show that the colonies are not always circular. Some microorganisms tend to form large flat colonies as it can be seen in Fig 4. In this case, I plated some of my kefir culture on a Sabouraud agar plate. You can even observe some yellowish colonies. Colonies are not always white or beige either. Not only can you choose different kind of agar media but also add some dyes for further characterization. One such example is shown in Fig 5. In this case bromocresol green is added to differentiate between microorganisms that can grow as white colonies and such as green ones. The color differences suggest that there are at least two different kinds of microorganisms on the plate shown in Fig 5.

Fig 5: Jolly Pumpkin’s Madrugada Obscura dregs on bromocresol green Sabouraud agar

Q: How do you determine the morphology of a colony?

First you need a pure culture of the microorganism. This is important because the morphology can differ if other microorganisms are in the same colony. The morphology can even be different on other agar media. Lets assume you want to describe the morphology of a pure brewers yeast (Saccharomyces cerevisiae). The first thing to do is streaking the yeast on a suitable agar media with a dilution streak and incubate the plate until colonies arise like shown in Fig 6. Sabouraud is a typical agar media for Saccharomyces and other yeasts. Malt agar media works as well.

Fig 6: Wyeast’s 2112 California Lager on Sabouraud Agar plate

In case of Fig 6, I streaked some of Wyeast’s 2112 California Lager yeast on a plate to check the purity. Now what about the morphology? Lets take a single colony and describe the following characteristics: form, margin, elevation (shape of the colony from the side), size, texture, appearance, pigmentation, opacity. The following descriptions are just an example.

Fig 7: from: http://commons.wikimedia.org/wiki/File:Bacterial_colony_morphology.png#filelinks; (Adapted and redrawn from Seeley, HW & Vandemark, PJ (1962) Microbes In Action: A laboratory manual of microbiology. WH Freeman (San Francisco, London) by user Ewen)

One might describe the colonies shown in Fig 6 as following:

Margins: Entire
Form: Circular
Elevation: Convex
Surface: Smooth
Opacity: Not transparent, shiny
Color: Off-white

That is what you can expect when you streak a yeast colony on a Sabouraud plate. The morphology of Saccharomyces is very similar on malt agar. Maybe some of you observed that there are yet some other different colonies on the plate in Fig 6. There were some impurities in this yeast sample as expected in the first place.

Q: Is the morphology of a given microorganism always the same?

Unfortunately not. The morphology of colonies can depend on the type of agar media used, if oxygen is present, nutrients, vitality, pH-levels, incubation time, other microorganisms present… Just keep in mind that a morphology description is not universal. If you encounter a morphology description of a specific microorganism, always check the type of agar media used and the conditions how the plates were incubated.

Q: Is it possible to differentiate between Saccharomyces and Brettanomyces based on morphology?

One of the most simple tricks to differentiate between the two yeasts is the incubation time. Saccharomyces colonies arise relatively quickly (within few days). Brettanomyces grow much slower (days to weeks). The second trick is to use a microscope and have a look at the different colonies. A third one might be (haven’t tried that one yet) to inhibit the growth of Saccharomyces by adding some growth inhibiting substances. Differentiating those two yeasts based on morphology is not that easy in my opinion.

Q: Is it possible to differentiate between top and bottom fermenting yeasts or even yeast strains based on colony morphologies?

As far as I know and from my experiences, differentiating between bottom and top fermenting yeasts base on colony morphologies is not possible. And it is not possible as well to differentiate between different yeast strains as well. Although I encountered some different morphologies for wheat strains at one point. However, I would not do any strain differentiation based on morphologies.

I hope there were some useful information in this post to give you a better understanding of agar media cultivation. Agar media are a very powerful tool in microbiology and is also widely used in breweries to check for impurities in beer or water. Understanding the concept of colony morphologies is therefore very important to get the most information out of agar media cultivation. And know about some limitations of the method as well.

# A glimpse into yeast growth kinetic models

Eureka, science post! Some math, model building and biology: I would like to start talking about yeast growth kinetic models today. In general, growth kinetics describes how different conditions (substrate, oxygen amount, metabolism products, inoculation rate, growth inhibitors etc) influence the growth of a microorganism in a time dependent manner. At the end one can construct mathematical models to describe the growth behaviour. In most cases one begins with experiments with fixed conditions and the growth of the organism is observed over time. A next experiment can be conducted with the change of one condition and the growth over time is observed once again. And so forth.

All these kinetic models can be very powerful tools. Not only can one improve for example the efficiency of yeast propagation but also investigate the effect of different parameters such as the inoculation rate of yeast, the substrate concentration, oxygen- or ethanol concentration on yeast growth. One application for growth kinetics could be to calculate yeast propagation like done in other yeast growth calculators for homebrewers.

This post is a general introduction about yeast kinetics and I would like to show you how one can get from experimental data to a simple growth kinetic model. Future posts will go into more detail and I would like to give a general introduction first.

# Exponential growth of microorganism

One of the most basic models in mathematical biology is the exponential growth equation for microorganisms. This equation can be used to calculate the amount of microorganisms or biomass (X) after a certain amount of time (t). Biomass can be the physical mass of the microorganisms, the cell concentration or any other measurement related to specify how many microorganisms there are (like optical density). I will stick to the physical mass of microorganisms as biomass in this post. In the exponential growth equation model no inhibition or substrate limitation is included. Substrate by the way is a term for any food source for the microorganisms such as glucose, maltose etc.

$X_t = X_{t0} \cdot e^{\mu_{max} \cdot t - t_0}$

• Xt is the biomass concentration at the time point t [g L-1]
• X0 is the biomass concentration at the time point zero [g L-1]
• µmax is the maximum specific growth rate [h-1]
• t Time [h]
• t0 Time where the experiment begins. Normally zero [h]

Lets make an example. Lets say you have a 1 L yeast starter (with indefinite amount of substrate) and add 10 g of yeast (X0) at the beginning (t0). You might ask yourself how many yeast cells you have after waiting for 90 min. The only thing you might not know is the specific growth rate (µmax). These coefficients can be looked up and for yeast µmax is somewhere around 0.5 h-1. My solution for this question would be 21.2 g L-1. So roughly double the amount.

You can even calculate the doubling time (tD) after changing the previous equation (doubling time specifies the time needed to double the initial amount of microorganisms):

$ln(\frac{X_t}{X_{t0}}) = \mu_{max} \cdot t$

By the way, the equation above will be important later on to get µmax. Moving on, at the time of doubling (tD), Xt equals 2 times Xt0:

$ln(\frac{2 X_{t0}}{X_{t0}}) = \mu_{max} \cdot t_D$

$ln(2) = \mu_{max} \cdot t_D$

$t_D = \frac{ln(2)}{\mu_{max}}$

Common doubling times for Saccharomyces cerevisiae are around 90 min [1]. This gives you a µmax of roughly 0.5 h-1.

In the exponential growth equation model substrate limitations are not included and therefore makes it not that useful to fully describe the growth behaviour if you want to investigate the effect of the substrate concentration itself. Let go to the next model.

# Monod equation for biomass

A very widely used model to describe the growth of microorganisms is the Monod model. The whole model is based on the Michaelis-Menten equation widely used in enzyme kinetics. I don’t want to go into further details here how one came up with these equations. In the end, the Monod model is again a simplified version of reality (like every model is). The first equation for the Monod model is:

$\mu = \mu_{max} \frac{S}{K_S + S}$

• µ is known as the specific growth rate [h-1]
• µmax is the maximum specific growth rate [h-1]
• S is the substrate concentration [g L-1]
• KS is the substrate saturation constant [g L-1]

We already know µmax, S is just the substrate concentration. For example the amount of dry malt extract you use for your yeast starter. Or glucose or whatever you are interested in. Just keep in mind that µmax depends on the used substrate. The definition of µ is:

$\mu = \frac{dX}{dt} \cdot \frac{1}{X}$

• µ is known as the specific growth rate [h-1]
• X is the biomass concentration [g L-1]
• dX is the change of the biomass concentration [g L-1]
• dt is the change of time when dX happens [h]

and depends on the change of biomass within an indefinite amount of time (dt). You could therefore write the Monod equation like:

$\frac{dX}{dt}= \mu_{max} \frac{S}{K_S + S} \cdot X$

and you already have your first differential equation for your model to describe the change of biomass in a time dependent manner.

This leaves KS. This is similar to the Michaelis constant Km in the Michaelis-Menten equation. In the case of the Michaelis-Menten equation, Km describes the substrate concentration at which the enzyme reaction is equal to half of the maximum speed. In the case of Monod, KS describes the substrate concentration (therefore S as index) where you have half of µmax. This is all so far for the first part of Monod.

# Monod equation for substrate

In the previous section, I introduced the basic Monod equation which can be used to describe the change of biomass over time. Next we like to include the change of substrate. In case of yeast one might be interested to investigate the behaviour of dry malt extract. For that we have to introduce another coefficient:

$Y_{X/S} = \frac{\frac{dX}{dt}} {\frac{dS}{dt}}$

• YX/S Biomass on substrate yield coefficient [gX gS-1]
• dX is the change of the biomass concentration [g L-1]
• dS is the change of the substrate concentration [g L-1]
• dt is the change of time when dX and dS happen [h]

YX/S simply defines how much biomass you can get from substrate (thus the index X and S). For example how much biomass you get from one gram of substrate. Looking at the previous equations, you can already see how to get a differential equation for the substrate:

$\frac{dS}{dt} = - \frac{\mu_{max} \frac{S}{K_S + S} \cdot X}{Y_{X/S}}$

Per definition, the equation is multiplied by minus one because the slope of dS over dt is negative (substrate is metabolized and therefore only decreases).

This leaves us to look at product kinetics. In case of yeast, one might be interested to describe the production of ethanol as a product. This is very similar to the substrate shown above. In this case you need another yield factor:

$Y_{P/X} = \frac{\frac{dP}{dt}}{\frac{dX}{dt}}$

• YP/X Product on biomass yield coefficient [gP gX-1]
• dP is the change of the product concentration [g L-1]
• dX is the change of the biomass concentration [g L-1]
• dt is the change of time when dX and dP happen [h]

In this case the product depends on the biomass and not on the substrate. Once again one can write down the differential equation for the product formation:

$\frac{dP}{dt} = \mu_{max}\frac{S}{K_S + S} \cdot X \cdot Y_{P/X}$

Are you still reading? Yes? You must either be very interested and/or be a math geek like me… I would like to stop with the introduction here and discuss other things such as inhibition etc in a future post. I now would like to share some information how you could/can get the coefficients for the equations above from empirical data.

# Coefficient determination from experimental data

All the data below is from a S. cerevisiae batch cultivation I cultivated during my undergraduate studies. I don’t want to get much into detail here but some information to understand the values you get afterwards. The batch cultivation was done in a 16 L reactor (4.2 gal) under sterile conditions and glucose as the only carbon source (substrate). All the values below therefore are for glucose only.

During the cultivation (8 h), every 30 min the optical density (OD) and glucose concentration was measured and every 60 min the dry mass was determined. The optical density is another measurement method to get an idea about the yeast concentration. The glucose was measured enzymatically and the dry mass was determined by filtration, drying of the filter paper and finally determining the mass of yeast on the filter paper.

# µmax determination based on dry mass

As previously mentioned, the following equation can be used to get the µmax value by plotting the logarithmic ratio of the biomass against the time (t). This should give you a linear function with the slope µmax as shown in Fig 1:

$ln(\frac{X_t}{X_{t0}}) = \mu_{max} \cdot t$

Fig 1: Log dry mass ratio against time. µmax determined from slope of linear fit function

One can easily see that the amount of yeast grew steadily up to the time point of six hours where the yeast concentration stayed the same (Fig 1). After six-hour the whole glucose was already metabolized (not shown) and no further yeast growth could be observed (Fig 1). The linear fit function therefore only makes sense between time point zero and six hours. The slope of the linear fit function was 0.43 h-1. Please remember, this µmax value is for the dry mass and is close to known values [2].

Lets quickly calculate the doubling time for this µmax. This give you a doubling time of roughly 97 min. Not that far away from the 90 min stated in source [1].

# µmax determination based on optical density (OD)

The same can be done for the OD (Fig 2).

Fig 2: Log OD ratio against time. µmax determined from slope of linear fit function

Once again after five to six hours, the yeast growth came to a stop (Fig 2). This time the slope of the linear fit function was 0.53 h-1. A nice example that not every µmax is the same. One has to be very careful how the specific µmax was determined. Either based on the optical density, dry mass weight or even the cell count.

# Biomass on substrate yield coefficient (YX/S)

To determine the yield coefficient, one can plot the measured yeast dry mass or OD against the measured glucose values and use the linear slope to determine the yield factors (Fig 3).

Fig 3: Determine the biomass to glucose yield factor

The yield factor based on the dry mass was -0.5 g g-1, and -0.1 g g-1 for the OD. Known yield factors for yeast based on glucose and dry mass are between -0.3 and – 0.5 g g-1 [3].

# Building growth models

Now its time to put all the equations and values into a model. The only coefficient not known so far is KS. This value is hard to determine and the easiest way to get this value is by iterative approaches. You simply use the model to get KS. I use Berkley Madonna for this purpose. What you have to do is input all the different differential equations, the measured values and run an iterative algorithm to let the model function approximate the measured values. If you do this the right way you might get graphs like below (Fig 4).

Fig 4: Yeast kinetic model 1, explanation in text below

In this case the substrate concentration (S) and the yeast concentration (X) are plotted against time. In addition, the black dots correspond to the measured glucose concentrations. I included the values I used for the different coefficients in the graph as well. Unfortunately, one cannot fit the measured glucose concentration curve with the numerical values of the determined coefficients. I therefore had to use slightly different values for the yield coefficient (in this case 0.3 g g-1) and 0.51 h-1 for µmax. The differences between the numerical values of the parameters might be due to measuring inaccuracies for the glucose concentration and/or yeast concentration. In the end, one can determine KS to be around 0.031 g L-1.

You now can use the model to investigate how the different conditions affect the growth of the yeast. For instance different substrate conditions or inoculation rates. All this can be useful to understand the behaviour of yeast growth under different conditions. However, one has to keep in mind that all this is based on a simplified model and it does not have to represent reality. Model building is sometimes hard work because iteration processes might get stuck and lead to wrong results (such as negative substrate concentrations).

# Summary

Growth kinetics models can be used to describe the growth of microorganisms. Because biological systems can hardly be approximated by simple fit functions, more sophisticated methods need to be applied. Such as the models described in this post.

# Outlook

This was just a basic introduction about growth kinetics and models. Future posts will go into more details covering additions to the basic Monod model. The next post concerning growth kinetics will be about Brettanomyces growth and is a nice organism to introduce inhibition effects.

I would like to do some small-scale experiments with yeast propagation and determine the individual model-parameters in the future. The resulting models therefore might be useful to approximate yeast starters under different conditions. Just be patient, I am currently really busy with my real scientific work. Thanks for reading and comment if you like. Cheers!

# Bibliography

[1] : http://dbb.urmc.rochester.edu/labs/sherman_f/yeast/4.html, “An Introduction to the Genetics and Molecular Biology of the Yeast Saccharomyces cerevisiae” (2012)
[2] : http://www.atcc.org (2012)
[3] : B. Sonnleitner, Lecture slides Bioprozesstechnik 1, ZHAW Wädenswil, 2010

# Saccharomyces bromocresol green screen

Eureka, I would like to share my latest plating results with you. You might know that I am very interested in isolating any kind of wild yeasts from commercial sour beers. The most difficult task in this whole isolation process is to differentiate normal Saccharomyces cerevisiae colonies from other yeast species such as Brettanomyces.

Previous studies to develop new kind of agar media to detect Brettanomyces in wine samples showed bromocresol green to be a useful indicator to detect acid producing Brettanomyces strains [Rodrigues et al., 2001; Couto et al., 2005, EP 1185686 A1]. In this case, bromocresol green acts as a pH-indicator and turns yellow in the presence of acid which is produced by some Brettanomyces species. The authors further added cycloheximide to the media to prevent any growth of Saccharomyces. Concluding from the previously cited publications an addition of cycloheximide to agar media should already be enough to differentiate between Saccharomyces and Brettanomyces colonies by simply inhibiting the growth of Saccharomyces. Further antibiotics could be added to prevent the growth of bacteria.

Other studies showed that bromocresol green alone can be used to differentiate between the two yeasts in absence of antibiotics [Yakobson, 2010]. In addition, bromocresol can diffuse into yeast colonies and form green colonies due to the accumulation of the dye [Yakobson, 2010]. However, some Brettanomyces strains seem to be able to form white colonies again. This has been shown in other experiments as well [Rodriguez, 2012; BKYeast, 2012]. Yakobson mentions that the dye gets actively metabolized by Brettanomyces and hence the white colonies again. Unfortunately, I could not find any source investigating how exactly the dye get metabolized. Yakobson further mentions that some Saccharomyces strains can form white colonies as well which would make it even more difficult to differentiate between Saccharomyces and Brettanomyces.

The aim of this study was to screen different Saccharomyces strains for their ability to form white colonies on bromocresol green containing agar.

# Material

• Sabouraud agar 4% glucose, Art. X932.1, Roth
• Bromocresol green sodium salt, Art. KK18.1, Roth
• Saccharomyces strains from Wyeast and White Labs

19 different Saccharomyces strains including one Saccharomyces mixture (WY3056) were plated on Sabouraud agar containing bromocresol green. Bromocresol green was added as aqueous, sterilized solution to the sterilized Sabouraud agar until the agar turned blue. The plates were incubated at room temperature at a dark place until colonies were visible. A control was included (no yeast streaked) to observe any color changes of the agar due to environmental effects (photo bleaching, oxidation, decay etc). The following yeast strains were used for this screen.

 Number Product name WY1010 American Wheat WY1056 American Ale WY1084 Irish Ale WY1728 Scottish Ale WY1762 Belgian Abbey II WY2112 California Lager WY2278 Czech Lager WY2487 Helle Bock WY3056 Bavarian Wheat Blend WY3068 Weihenstephan WY3333 German Wheat WY3522 Belgian Ardennes WY3638 Bavarian Wheat WY3711 French Saison WY3726 Farmhouse Ale WY3864 Canadian/Belgian Ale WY3942 Belgian Wheat WY3944 Belgian Wit WLP002 English Ale Control N/A

# Results Part 1

Colonies were visible after four days of incubation (Fig 1-5). The control showed no colony formation and the agar showed no color change. The colors of the agar were compared with the control.

Fig 1: Yeasts on bromocresol agar after four days. Left: WY1762, Top: WY3522, Right: WY3864; Bottom: Control

Fig 2: Yeasts on bromocresol agar after four days. Left: WY3942, Top: WY3944, Right: WY3726; Bottom: WY3711

Fig 3: Yeasts on bromocresol agar after four days. Left: WY2487, Top: WY2112, Right: WY2278; Bottom: WY1010

Fig 4: Yeasts on bromocresol agar after four days. Left: WY3638, Top: WY3068, Right: WY3333; Bottom: WY3056

Fig 5: Yeasts on bromocresol agar after four days. Left: WLP002, Top: WY1056, Right: WY1728; Bottom: WY1084

Some Saccharomyces strains were able to change the color of the agar from green-blue to yellow. Only two yeast strains, WY3333 German Wheat and WY3726 Farmhouse Ale, grew as white colonies on bromocresol green agar after four days. This already is proof that some strains indeed can grow as white colonies on bromocresol green. The plates were further incubated and after a total of twelve days, the color of the colonies were evaluated for a second time (Fig 6-10). Sorry for the bad quality of the pictures.

Fig 6: Yeasts on bromocresol agar after 12 days. Left: WY1762, Bottom: WY3522, Right: WY3864; Top: Control

Fig 7: Yeasts on bromocresol agar after 12 days. Left: WY3942, Top: WY3944, Right: WY3726; Bottom: WY3711

Fig 8: Yeasts on bromocresol agar after 12 days. Left: WY2487, Top: WY2112, Right: WY2278; Bottom: WY1010

Fig 9: Yeasts on bromocresol agar after 12 days. Left: WY3638, Top: WY3068, Right: WY3333; Bottom: WY3056

Fig 10: Yeasts on bromocresol agar after 12 days. Left: WLP002, Top: WY1056, Right: WY1728; Bottom: WY1084

One could observe that some of the colonies now have white edges and a green centre. All these colonies were still counted as green colonies.

This time less yeast strains turned the agar to a yellow color because the control agar lost a lot of its blue color. The global decrease of the blue color in the agar might originate from diffusion of acids secreted by yeasts that turned the agar yellow. Or due to the diffusion of the dye into the colonies. Further on to the white yeast colonies. WY3333 German Wheat and WY3726 Farmhouse Ale still grew in white colonies. In addition, WY3864 Canadian/Belgian Ale and WLP002 English Ale now grew as white colonies as well. One might expect further yeast strains to form white colonies with a prolonged incubation time because a lot of the colonies already have white edges and a remaining green centre.

After 17 days of incubation, the colonies looked as shown below (Fig 11).

Fig 11: Yeasts on bromocresol agar after 17 days. Left/bottom: WY2487, Left/Top: WY2112, Right/Top: WY2278; Right/Bottom: WY1010

A lot of yeast colonies now turned into white colonies as expected (Fig 12). The color was now evaluated by looking at the entire colonies visible for a particular strain. If more than 50% of the colonies were white, the yeast was counted as white. Like the WY2278 Czech Lager shown in Fig 11. On the other hand, all the other yeasts shown in Fig 11 were counted as green like the WY3711 French Saison in Fig 12.

Fig 12:WY3711 French Saison colonies after 17 days of incubation

After 17 days of incubation, only seven out of the 19 screened yeasts still had green colonies. All the other ones turned white in the meantime. To put it in numbers. After 4 days 2/19, after 12 days 4/19 and after 17 days of incubation 12/19 yeast strains formed white colonies (Fig 13). This clearly shows a time dependency.

Fig 13: Yeast screen results on heavily stained bromocresol green agar

# Results Part 2

The blue color in the agar plates (Fig 1-5) was quite heavy and to test whether a lower concentration of bromocresol green in the agar leads to the same results as discussed above, a second experiment was conducting by streaking the exact same yeast strains on some Sabouraud agar containing bromocresol green. This time a lower concentration of bromocresol green was used.

Quantification of the color after three days of incubation (Fig 14-18):

Fig 14: Yeasts on bromocresol agar after three days. Left: WY3864, Top: Control, Right: WY1762; Bottom: WY3522

Fig 15: Yeasts on bromocresol agar after three days. Left: WY3942, Top: WY3944, Right: WY3726; Bottom: WY3711

Fig 16: Yeasts on bromocresol agar after three days. Left: WY2112, Top: WY2278, Right: WY1010; Bottom: WY2487

Fig 17: Yeasts on bromocresol agar after three days. Left: WY3638, Top: WY3068, Right: WY3333; Bottom: WY3056

Fig 18: Yeasts on bromocresol agar after three days. Left: WLP002, Top: WY1056, Right: WY1728; Bottom: WY1084

Yet again some colonies grew as white colonies and others grew as green ones (Fig 19). Comparing the results with the one concluded from the first experiment, WY3726 Farmhouse Ale, WLP002 English and WY3864 Canadian/Belgian Ale showed white colonies. In contradiction with the first experiment are the color morphologies of WY3333 German Wheat, WY1728 Scottish Ale and WY3711 French Saison. WY3333 grew as white colonies in the first experiment and as green ones in the second one. On the other hand, WY3711 and WY1728 grew as white colonies in the second experiment.

Fig 19: Closer look at Fig 15

The colours were again determined after further incubation. Agar plates shown after 12 days of incubation (Fig 20-24).

Fig 20: Yeasts on bromocresol agar after 12 days. Left: WY3864, Top: Control, Right: WY1762; Bottom: WY3522

Fig 21: Yeasts on bromocresol agar after 12 days. Left: WY3942, Top: WY3944, Right: WY3726; Bottom: WY3711

Fig 22: Yeasts on bromocresol agar after 12 days. Left: WY2112, Top: WY2278, Right: WY1010; Bottom: WY2487

Fig 23: Yeasts on bromocresol agar after 12 days. Left: WY3638, Top: WY3068, Right: WY3333; Bottom: WY3056

Fig 24: Yeasts on bromocresol agar after 12 days. Left: WLP002, Top: WY1056, Right: WY1728; Bottom: WY1084

Twelve days of incubation and all the yeast strains have the same color like a few days ago. The plates were further incubated and a final color determination was conducted after 17 days (not shown).

The results of the second run are summarized in Fig 25. WY1010 American Wheat, WY1084 Irish Ale, WY1762 Belgian Abbey II, WY2112 California Lager, WY2278 Czech Lager, WY3068 Weihenstephan and WY3942 Belgian Wheat all had white colonies after 17 days (Fig 25). 13/19 yeast strains grew as white colonies after 17 days of incubation (Fig 25).

Fig 25: Yeast screen results on light-stained bromocresol green agar

# Discussion

Comparing the two experiments, some strains such as WY1010 American Wheat, WY1728 Scottish Ale, WY2112 California Lager, WY3711 French Saison and WY3942 Belgian Wheat only grew in white colonies after 17 days on the light stained agar media and not the heavy stained one (Fig 26). WY3056 Bavarian Wheat Blend, WY3522 Belgian Ardennes and WY3638 Bavarian Wheat grew as white colonies on heavily stained agar but as green ones on lightly stained agar media (Fig 26). This might be an indicator that the bromocresol green concentration might influence the color change as well.

Fig 26: Differences between the two experiments

As a general trend, the different yeast strains seem to form white colonies after further incubation. However, two strains (WY3333 German Wheat and WY3726 Farmhouse Ale) grew on heavily stained agar as white colonies from very early on (Fig 13) and four additional ones on lightly stained agar (Fig 25). Yakobson states on his website that Wit yeasts can metabolize bromocresol green (http://www.brettanomycesproject.com/2009/03/wln-agar-medium/). In this screen the Wit strain from Wyeast (WY3944) did not grew as white colonies in both experiments (Fig 13, 25). Not even after 17 days of incubation.

Some words about the color of the agar media. Fig 1 to 5 are nice examples to show that the color of the bromocresol containing media changes its color from green to yellow. In both experiments, the color of the control agar turned to a yellow color as well. The plates were stored at a dark place to prevent any influence of light (photobleaching effects). The change in color might be due to secretion of acids (bromocresol changes color at lower pH to yellow), due to a take-up of the dye by the yeast cells like stated by Yakobson in case of Brettanomyces. Another possibility might be the stability of bromocresol green itself. If one imagines bromocresol green to be a relatively unstable molecule, the loss of the green color might be due to the depletion of the dye. Yakobson further mentions that Brettanomyces can even metabolize the dye and therefore grow as white colonies. All the cells not able to metabolize the dye remain as green colonies. Unfortunately, I could not find any evidence for this statement showing that Brettanomyces really metabolize the dye. Nor any evidence that Saccharomyces can do it. Maybe the cytoplasm of Brettanomyces cells have a lower pH and therefore turn the dye from green to yellow. There might even be some truth about this hypothesis since some Brettanomyces strains are known to secrete acetic acid under aerobic conditions. It is therefore not clear to me why/how the colonies turn from green to yellow.

I would like to discuss bromocresol green as a useful tool to differentiate between Brettanomyces and Saccharomyces. Although I did not show any Brettanomyces colonies here, the bromocresol screen strongly suggests that some Saccharomyces strains can grow as white colonies on bromocresol green containing agar media. This makes a differentiation already a bit harder. In addition, a majority of Saccharomyces yeast strains appear as white colonies after a longer incubation period. BKYeast came to the conclusion that differentiation based on bromocresol green might only be possible in a short time frame in mixed cultures (Saccharomyces and Brettanomyces grow on the same plate). The results from these experiments show that even in pure cultures, and in absence of Brettanomyces, a lot of the Saccharomyces strains tested turned from green to white within a short period of time. All these results strongly suggest that any differentiation solely based on bromocresol green might only be useful in a short period of time.

# Summary

Bromocresol screen is a widely used differentiating dye to differentiate between Saccharomyces and Brettanomyces. Brettanomyces known for their capability to grow as white colonies while Saccharomyces grow as white ones. It has been reported that some Saccharomyces strains grow as white colonies as well and therefore making a differentiation more difficult [Yakobson, 2010]. Screening different Saccharomyces cerevisiae strains on bromocresol green containing Sabouraud agar revealed some strains capable of growing as white colonies from the very beginning where the majority of yeast strains grew as green ones. Therefore showing that indeed some yeast strains can grow as white colonies. After further incubation, the majority of the yeast strains turned from green to white coloured colonies. There seems to be a general trend for Saccharomyces cerevisiae strains to form white colonies after extended incubation times. However the reason for this observation is not clear at this point as well as the mechanism leading to the observed change in color. It can’t be excluded that different sources like instability of bromocresol green itself or any environmental factor lets the colonies turn from green to yellow.

Due to these observations, bromocresol green as a tool to differentiate between Brettanomyces (known to grow as white colonies) and Saccharomyces might only work within a small time frame. This has been previously observed by BKYeast as well.

# Outlook

Brettanomyces bromocresol green screen similar to the one shown here for Saccharomyces. In addition, try to grow Brettanomyces anaerobically to test whether the colonies grow as white or green ones (acid theory mentioned in the discussion).

# References:

I am open to any discussions and feedback concerning this experiment. Thank you for reading.

# Insight into the Brettanomyces Mitochondrial genome

Eureka, I have to mention first that this is a very scientific biochemistry, bioinformatics post about Brettanomyces. However, I hope those of you with less biology experience can follow as well or at least get the idea of the main messages. This post is mainly about an insight into the mitochondrial genome of two Brettanomyces species and comparing those sequences with Saccharomyces.

I always wondered how similar the genome sequence of a Saccharomyces cerevisiae and Dekkera/Brettanomyces yeasts are. To do such comparisons the DNA sequences have to be published first or at least sequenced. I am not yet through all the publications about the Brettanomyces genome sequencing and therefore don’t know yet if any complete genome of Brettanomyces/Dekkera is available at the moment. More about the yeast genomes in future posts.

I would like to start with a quick introduction about the whole topic to give those without a biology background a chance to understand the following points. This post is about comparing different DNA sequences from different yeasts. You might know that every living organism contains DNA which can be seen as the manual of the cell. The DNA encodes nearly everything the cell needs to work such as proteins, different RNAs etc. The whole DNA within a cell is commonly called genome. The DNA consists of four bases: Adenine, guanine, cytosine and thymine. In the DNA every information is encrypted with these four bases. The code to decipher the information from the four bases code into a protein is called the genetic code. If the genetic code is known you can encode a DNA sequence into proteins. Or vice versa. Before one can encode a DNA sequence the sequence has to be known. And this is the process called DNA sequencing. By DNA sequencing you determine the sequence of the four bases in the DNA. Knowing DNA sequences is very important for modern biology to for example understand certain diseases. On the other hand, DNA sequences are in general very specific for every living organism. This can be used to detect certain organisms. I hope this is enough information for the beginning.

One disadvantage of sequencing genomes of yeasts is their size. Sequencing big genomes is always challenging. Before modern techniques such as shotgun sequencing came widely available the only way of sequencing large genomes was really time-consuming and thus really expensive. This is different today and a lot of people around the world are working on sequencing projects. Me included.

To compare any genomic sequences or get any information about the evolutionary relationship of Saccharomyces and Brettanomyces, one has to look at the DNA of the two yeasts and compare them. And since I don’t know yet if the full genome sequence of at least one Dekkera/Brettanomyces strain is sequenced one has to look at a different DNA. And that’s where the mitochondrial genome comes into play. Mitochondria are the organelles in the cell responsible for several pathways such as supplying the cell with energy. And mitochondria have their own DNA, called mitochondrial DNA (mtDNA) because mitochondria originate from bacteria cells (see endosymbiotic theory). To summarize this theory, some time ago a cell incorporated a bacteria cell and the incorporated bacteria cell lived on within the first one and became the mitochondria. And since the mitochondria was a bacteria cell with DNA, the DNA still exists in some part within the mitochondria. The whole amount of this mtDNA is then called the mitochondrial genome. Because mtDNA are relatively short compared with the genomic DNA of a yeast, sequencing mtDNA is relatively easy. The first sequencing of a mtDNA sequence of D. bruxellensis and B. custersianus has been completed by E. Procházka et al in 2010 [Procházka et al, 2010]. All the data below is from this publication.

Since Saccharomyces cerevisia is an eukaryotic model organism for scientists, the whole genome of this yeast is already sequenced. Including the mtDNA. The mitochondrial genome sequence has been published and can be found here under the accession number AJ011856. For D. bruxellensis (strain CBS 2499) the sequence is deposited with the accession number NC_013147. And for the B. custerianus strain (CBS 4805) with the accession number GQ354525. If you look at one of the deposited genomes you can see the sequence at the bottom of the entry.

Lets begin with a look at the mtDNA of Saccharomyces cerevisiae. One can see that the mtDNA is circular and 85779 bp long (Fig 1). All the red arrows represent a specific gene encoded on the DNA. I only included the genes because with all the other annotated stuff such as tRNA etc the picture would simply be unreadable. One can observe that it seems that all the genes are facing in the same direction. This simply means that only one strand of the DNA is used for coding. (DNA is double stranded).

Fig 1: mtDNA Saccharomyces cerevisiae

Moving on to D. bruxellensis. Again a circular mtDNA (Fig 2). By the way, the publication mentioned above was the first to demonstrate that the mtDNA in Brettanomyces is circular. This mtDNA is 76453 bp long and includes a lot of genes as well. However, some genes face a different direction than others. The mitochondrial genome in D. bruxellensis therefore uses both strands as coding strands. This is already different compared to S. cerevisiae in Fig 1.

Fig 2: mtDNA D. bruxellensis

And at last a quick look at the mtDNA in B. custersianus (Fig 3). Yet again a circular mtDNA with a length of 30058 bp. This is much shorter than in S. cerevisiae and D. bruxellensis. And yet again, the genes are read on only one strand.

Fig 3: mtDNA B. custersianus

Lets briefly summarize the first few observations and lets compare them. All the mtDNA genomes are circular. The sizes in S. cerevisiae and D. bruxellensis are more or less the same. The mtDNA in B. custersianus though is significantly shorter than the other two yeasts. On the other hand the genes in both S. cerevisiae and B. custersianus are one single strand and genes in D. bruxellensis on both.

All these observations already tell me that these three yeasts are really not the same based on their mitochondrial genomic setup. One might argue about the size differences but the different gene orientation is quite remarkable in my opinion.

Moving on with further comparisons. Below is a table with the number of genes encoded in the mitochondrial genomes in all three yeasts and the number of tRNAs (transfer RNA). If you don’t know what tRNAs are just don’t bother. I will not get into any details about these tRNAs in this post.

 S. cerevisiae D. bruxellensis B. custersianus Genes (including tRNA) 42 46 47 tRNA 24 25 25 Genes (without tRNA) 18 21 22

By just looking at the number of genes one might already tell that there seems to be a difference in the number of genes without tRNAs between S. cerevisiae and Brettanomyces/Dekkera. All the mitochondria seem to have roughly the same number of tRNAs. I will not get into further detail about the tRNAs here. If you need further information please have a look at the original publication. I would like to talk about the differences in genes instead.

Lets have a closer look at some of the genes encoded in the mitochondrial DNA in the three yeasts. Please keep in mind that over 99% of the proteins present in the mitochondria originate from the cytosol: Newly synthesized cytosolic proteins are transported from the cytosol across the outer membrane by the TOM40:TOM70 complex. Thus the mitochondria DNA does not have to encode for a lot of proteins as it can easily be seen by looking at the table above.

The following genes are encoded within the mtDNA:

• Cytochrome oxidase subunits 1, 2, 3 (cox1, cox2, cox2)
• Apocytochrome b (cob)
• ATP-synthase subunits 6, 8, 9 (atp6, atp8, atp9)
• Mitochondrial small and large rRNAs (rns, rnl)
• RNAse P (rnpB)
• Mitochondrial subunit ribosomal protein 3 (rps3)

A lot of these proteins can be found in S. cerevisiae as well. But this does not mean the DNA sequences are the same though. More about that later on. Indeed there are some genes only encoded in the two Brettanomyces/Dekkera mtDNAs:

This is an enzyme complex (also known as respiratory chain complex I) and catalyzes the reaction of NADH to NAD+.

The next question to answer is if the genes found in both yeast species have the same sequences or not. I would like to look at one set of genes.

# Cytochrome c oxidase:

The cytochrome c oxidase complex consists of three different subunits, subunit 1 to 3. Cytochrome c oxidase catalyzes the reduction of oxygen to water and plays a very important part in the respiratory chain. Below is a table showing the length [bp] of the different genes.

 S. cerevisiae D. bruxellensis B. custersianus COX1: cytochrome c oxidase subunit 1 12884* 5348* 5436* COX2: cytochrome c oxidase subunit 2 756 738 744 COX3: cytochrome c oxidase subunit 3 810 810 810

Both yeasts have the three subunits encoded in their mitochondrial DNA. However, in D. bruxellensis subunit 1 has 3 exons, 4 exons in B. custersianus, and 8 exons in case of S. cerevisiae. The CDS (coding sequence) in D. bruxellensis and B. custersianus are 1629 bp and 1605 bp long in S. cerevisiae.

Comparing the two protein sequences of the Brettanomyces/Dekkera strains shows a pairwise identity of 85.1% (MAFFT alignment). For the other two subunits 2 and 3 in the two strains 88.8% for COX2, 81.9% for COX3.

Comparing COX1 from D. bruxellensis with S. cerevisiae shows a pairwise identity of 70.3%. COX1 from B. custersianus and S. cerevisiae are 70.5% identical.

Comparing COX2 from D. bruxellensis with S. cerevisiae shows a pairwise identity of 75.8% (Fig 4). COX2 from B. custersianus and S. cerevisiae are 73.9% identical.

Comparing COX3 from D. bruxellensis with S. cerevisiae shows a pairwise identity of 70.4%. COX3 from B. custersianus and S. cerevisiae are 70.1% identical.

Fig 4: Comparing COX2 from D. bruxellensis with S. cerevisiae shows a pairwise identity of 75.8%

This is a very nice example that all the three yeasts have the same protein function (cytochrome c oxidase) but the sequences are not the same and not even encoded the same way (different exons) or direction (as shown above). On the other hand not even the two Brettanomyces/Dekkera strains have the same sequences. Still the sequences are more identical in the two Brettanomyces/Dekkera strains than compared to S. cerevisiae.

Putting this all together, the three different yeast strains look very different at a molecular level (different size, using two strands as coding strands). The genes don’t even have the same exact sequences. In the end all the yeasts produce proteins with the same function. This is quite remarkable in my opinion. Another big difference between S. cerevisiae and Dekkera/Brettanomyces is the existence of the NADH dehydrogenase in the latter yeasts.

I hope this post was not too complicated and got you some ideas about the different genetic setup of Saccharomyces and Brettanomyces/Dekkera. In the end all the yeasts achieve the same but all with a different setup. This is simply remarkable and in my opinion a brilliant example how evolution impacts an organism. Not to say that even within the same species such as Brettanomyces/Dekkera two different strains (D. bruxellensis and B. custersianus) might have very different setups as well. This makes me wonder what a look at the genomic DNA might reveal…

I would like to end by mentioning that this is a nice example to show people what one can do by just looking at DNA sequences and why it is important in my opinion to sequence organisms in the first place. Such insights are not possible if no DNA sequences are available. Don’t expect the genomic Brettanomyces DNA insight soon. This will take me much longer to prepare because there is much more data to process…

# Bibliography

• E. Procházka, S. Poláková, J. Piskur and P. Sulo, 2010. Mitochondrial genome from the facultative yeast Dekkera bruxellensis contains the NADH dehydrogenase subunit gene. FEMS Yeast Res, 10, 545-557 (Pubmed)