Evaluate starter media to propagate Lactobacillus sp.

Welcome back everyone. Yes, I am still alive. Although my job in science absorbs lots of my spare time lately, I still find time now and then to brew on a semi-professional scale. Which unfortunately leaves yeast science and this blog at lower priorities. I still do yeast work at home but it all shifted to more practical applications like establishing and testing blends, evaluating yeast isolates and playing around with some full size wine barrels.

What I want to share today are the results of an evaluation experiment I performed a couple of months ago to look for MRS media alternatives to propagate lactobacillus at home. MRS media is kind of the golden standard used to propagate lactobacillus. It works very well but with the disadvantage of being a quite expensive media. I therefore tested a couple of cheaper alternative media and compared the growth/propagation efficiency with MRS. Please notice that the experiment and results are added in a rather short kind of way. That’s all from me now. Take out your pencils & notepads and start reading. Over&Out.

Goal of project

  1. Set up starter conditions and protocols to propagate Lactobacillus sp. to pitchable amounts
  2. Compare growth properties to MRS broth as reference
  3. Determine most efficient media to propagate Lactobacillus sp.

Material & Methods

The following media were tested:

  1. Lactobacillus media 1: 100% apple juice
  2. Lactobacillus media 2: 100% apple juice + CaCO3 (20 g L-1)
  3. Lactobacillus media 3: 100% apple juice + CaCO3 (20 g L-1) + yeast nutrients
  4. Lactobacillus media 4: 10°P DME
  5. Lactobacillus media 5: 10°P DME, 10% (v/v) apple juice
  6. Lactobacillus media 6: 10°P DME, 10% apple juice + CaCO3 (20 g L-1)
  7. Lactobacillus media 7: 10°P DME, 10% apple juice + CaCO3 (20 g L-1) + yeast nutrients
  8. MRS-Bouillon (as reference, CarlRoth prepared according to manual)

10°P DME starter and MRS media was autoclaved (15 min at 121°C) and mixed with additional components at room temperature. Pasteurized apple juice was used. Each media (50 mL in total) was inoculated with 1 mL of bacteria culture (Wyeast 5335 L. delbruecki/L. buchneri; Wyeast 5223 L. brevis). Propagation performed at room temperature (no shaking, no aeration).

To address the efficiency of the media, the culture densities were estimated based on microscope observations after 7 days of propagation.

Results

WY 5335 L. delbrueckii/L. buchneri:

  1. Media 1: None-few LAB cells (rod-shaped) visible
  2. Media 2: Yeast & circular bacteria cells visible (contamination)

    Ldelbrueckii_Med2_01

    Media 2: Yeast & circular bacteria cells visible (contamination)

  3. Media 3: Circular bacteria visible (very few LABs)

    Ldelbrueckii_Med3_01

    Media 3: Circular bacteria visible (very few LABs)

  4. Media 4: LAB visible

    Ldelbrueckii_Med4_01

    Media 4: LAB visible

  5. Media 5: LAB visible

    Ldelbrueckii_Med5_01

    Media 5: LAB visible

  6. Media 6: LAB visible

    Ldelbrueckii_Med6_01

    Media 6: LAB visible

  7. Media 7: Lots of LAB

    Ldelbrueckii_Med7_01

    Media 7: Lots of LAB

  8. MRS control: Lots of LAB maybe more than media 7

    Ldelbrueckii_MRS

    MRS control: Lots of LAB maybe more than media 7

WY 5223 L. brevis:

  1. Media 1: None-few LAB
  2. Media 2: Circular cells, few rod-shaped bacteria
  3. Media 3: Circular cells, few rod-shaped bacteria
  4. Media 4: LAB visible
  5. Media 5: LAB visible, more or less the same as for media 4
  6. Media 6: Lots of LAB

    Lbrevis_Med6_01

    Media 6: Lots of LAB

  7. Media 7: Lots of LAB, maybe same as media 6

    Lbrevis_Med7_01

    Media 7: Lots of LAB, maybe same as media 6

  8. MRS control: Lots of LAB, same as media 7

pH-measurements after propagation

Unfortunately, I was not able to measure the pH of the media prior to the propagation. Just received my fancy pH-meter a bit to late for that. Below the pH measurements of the media after propagation.

Media // L. delbrueckii // L. brevis
1 // 3.21 // 3.23
2 // 5.80 // 5.87
3 // 6.54 // 5.92
4 // 4.08 // 3.32
5 // 3.10 // 3.22
6 // 5.68 // 4.57
7 // 5.42 // 4.82
MRS // 4.18 // 4.44

Summary & Conclusions

  • LAB grow very well in MRS media (room temperature, no aeration)
  • Both LAB samples tested grew in various of the tested media. Apple juice, even in presence of other components, does not lead to optimal growth efficiencies compared with the MRS controls
  • Propagation in Lactobacillus Media 7 (10°P DME + 10% (v/v) apple juice + 2% (w/v) CaCO3 and yeast nutrients leads to growth efficiencies close to MRS media

In conclusion, growing/propagating LAB in Lactobacillus Media 7 seems to be the most efficient media tested in this series with results similar to MRS media.

Sugar composition of wort

Short one for today. I would like to share some information about the sugar composition of wort since I had to take this into consideration for an upcoming project I am preparing for publishing here soon (yes, its yeast related). Lets talk sugars today!

I am not sure how many homebrewers thought about the actual sugar composition of their wort before. And I am not speaking about fermentable and non-fermentable ones. The real composition like sucrose, maltose, glucose etc. The question now is why one might think about that problem in the first place. For example, if you are interested to know if a non-Saccharoymces yeast (capable of fermenting glucose only) can ferment something in a wort, you might need to know if glucose is even present in the first place (and this example is pretty close to the question I asked myself to eventually investigate the composition of sugars in wort).

The composition of sugars in wort has been addressed a couple of years ago and published in various papers. Like “Determination of the sugar composition of wort and beer by gas liquid chromatography” by Otter et al published in 1967 [get me to the paper]. I will not go into the scientific details as well as experimental setup of this paper but would like to discuss the results.

header_paper

Fig 1: Paper header

Otter et al determined the concentrations of six sugars (fructose, glucose, sucrose, maltose, maltotriose and maltotetraose) in 15 different worts with various OGs (ranging from 1.027 up to 1.093). I averaged the sugar compositions of the 15 samples as amount of sugar X relative to the total amount of sugar present in wort. Just to give me a rough idea. So don’t read too much into the numbers here. It’s about the ratio or more like sugar X is highly abundant or not. And yes, I thought about effects of grain bill composition, mash schedules, mash pH, you name it on the sugar composition. Getting a rough idea here.

test

Fig 2:Sugar composition w[%] of total sugar as average of 15 different worts

About half of the sugars present in wort is maltose (Fig 2). Followed by maltotriose and glucose. And some smaller amounts of fructose, sucrose and maltotetraose. Maltotetraose by the way is a dextrin and can be counted as non-fermentable. Standard Saccharomyces cerevisiae strains are capable of fermenting all the present sugars except maltotetraose. Which might explain why S. cerevisiae is the working horse of brewers. In summary, maltose makes up about half of the total sugars followed by glucose and maltotriose. And some minor amounts of fructose, sucrose and maltotetraose. I am actually surprised about the amount of glucose present in wort. I did not expect that at all.

So there you go. I will address the initial problem about a specific sugar metabolism of a non-Saccharomyces yeast in a future post including some empirical data. Stay tuned!

Brettanomyces Phylogenetic II & WLP Brett Trois crisis

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

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

alignment_38bretts_pyhlotree

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

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

text473

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

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

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

Fig 3: MSA B. naardenensis sequences & Pichia guilliermondii

Fig 3: MSA B. naardenensis sequences & Pichia guilliermondii

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

WLP Brettanomyces Trois ID crisis

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

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

ITS2_seq_MSA

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

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

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

ITS2_seq_MSA_extracted

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

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

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

phylo_tree_extracted_ITS2_seqs

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

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

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

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

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

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

Where do I work?

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

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

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

What about beer?

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

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

It’s time to vote for your favourite Eureka Yeast Lab Brett strain

Eureka, its time to announce some changes. First, I would like to announce that Eureka Brewing Yeast (EBY) strains will be replaced by Eureka Yeast Lab (EYL) strains as lots of people mixed up my strains with the ones offered by East Coast Yeast (ECY). I would like to apologize to Al Buck for the unwise abbreviation choice for my yeast strains. I furthermore hope that EYL is easier to distinguish from ECY products. The product numbers will stay the same but EBY will be changed to EYL.

Second, its been a while since Jeff and myself started the EBY/BBA Brettanomyces experiment and its now time to put some action into this story. Although the amount of data I got back from the experiment isn’t as great as expected, its time to prepare the release of two Brett strains. And now is the time to vote for your favourite strain. Or the one looking most promising to you. Only the two strains with the most votes will get released in a couple of weeks. So hurry up and spread the word. The poll will be closed on Monday, 25th of August.

The release will be first announced to the Google Group members at https://groups.google.com/d/forum/eureka-brewing-yeast and later on posted on my blog. Giving the members an advantage. If no vials remain after the Google Group shout-out, no release on my blow will follow. To join the group without a gmail account, simply write an email to eureka-brewing-yeast+subscribe@googlegroups.com to get subscribed. That’s it for today. To Brett biodiversity!

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
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Insight into the genome of Saccharomyces carlsbergensis

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

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

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

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

Loss of parental S. cerevisiae DNA in S. carlsbergensis

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

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

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

Chromosomal map of S. carlsbergensis

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

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

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

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

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

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

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

Bibliography

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