Brettanomyces Research Fermentation Photos

Thought I would include some photos of the primary fermentations to give some insight into the research project and show how I set up to observe 105 trial fermentations.

 

Matted & Glossy strains and WhiteLabs strains in a temperature controlled incubator

 

 

 

 

 

 

A close up with some of the fermentations containing Lactic acid

 

 

 

 

 

 

Matted & Glossy, Wyeast, and WhiteLabs strains with different pitch rates in a temperature controlled incubator

 

 

 

 

 

 

Preparing Wyeast strains before moving to a temperature controlled environment

 

 

 

 

 

 

Surface yeast or pellicle forming on WLP650 B. bruxellensis

 

 

 

 

 

 

Close up of the thin pellicle

Research Methodology – Brett Primary Fermentations

While performing the propagation trials, I prepared my research methodology for the primary fermentations, which would make up the bulk of my research. As previously mentioned all mini-fermentations were conducted with wort produced on the 2 hectolitre pilot system. The following details the experiments I put together in researching the characteristics of a Brettanomyces primary fermentation. My intent was to observe what compounds are produced during the fermentation, how those compare to a general ale, and also to observe a few known aroma compounds produced. The main aroma compound I wanted to observe was Ethyl Lactate, an ester produced from ethyl alcohol and Lactic acid. Ethyl lactate is believed to create a pineapple or tropical fruit aroma. By adding Lactic acid to the wort, pre-fermentation in varying amounts, I planned to observe the levels of Ethyl Lactate formed, and correlate it to the aroma found in the specific fermentation conducted.

First Tier: Pitching Rates
•    Trial fermentations conducted in duplicate in 2000mL Duran glass bottles. The dimensions were 135mm:200mm (diameter:height) or a ratio of 1:1.5 with a conical headspace of 20% volume.
•    1.8L (1,800mL) of standard (autoclaved) wort used with 10ppm dissolved oxygen diffused into the wort initially. (roughly 1ppm per °Plato)
•    Anaerobic fermentations were conducted by inserting rubber bungs air tight into the tops of the glass bottles and connecting rubber air tubing, allowing blow-off of Carbon dioxide gas into sterile water.
•    Fermentations conducted at 21-22°C (70-72°F) in temperature-controlled incubators.
•    Starting Plato of wort 12° (1.048 gravity)
•    Starting pH of 4.95 (±) .05

Primary pitching rate = 1.0×10^6cells/mL×12°P×1800mL=2.16×1010cells
•    When counting cells for determining amounts to be measured out to pitch, viability was also determined, recorded, and taken into account to get the actual amount of viable cells for pitching (shown in red). The following shows the calculation used to determine the volume of propagated cells needed within each trial fermentation.
•    B. claussenii (WLP645) 2.16×1010cells/6.59×108=32.77mL (vol. pitched into wort)
•    B. claussenii (WY5151) 2.16×1010cells/10.80×108=20.00mL (vol. pitched into wort)
•    B. bruxellensis (WLP650) 2.16×1010cells/6.64×108=32.53mL (vol. pitched into wort)
•    B. bruxellensis (WY5112) 2.16×1010cells/10.66×108=20.26mL (vol. pitched into wort)
•    B. lambicus (WLP653) 2.16×1010cells/5.79×108=37.30mL (vol. pitched into wort)
•    B. lambicus (WY5526) 2.16×1010cells/13.94×108=15.5mL (vol. pitched into wort)
•    B. matted (CMY-001) 2.16×1010cells/5.90×108=36.61mL (vol. pitched into wort)
•    B. glossy (CMY-002) 2.16×1010cells/8.18×108=26.40mL (vol. pitched into wort)

For Secondary and Tertiary pitch rates, 4 strains were chosen for observation in the trials.

Secondary pitching rate = .5×10^6cells/mL×12°P×1800mL=1.08×1010cells
•    B. claussenii (WY5151) 20.00mL/2=10.00mL (vol. pitched into wort)
•    B. lambicus (WY5526) 15.5mL/2=7.75mL (vol. pitched into wort)
•    B. matted (CMY-001) 36.61mL/2=18.30mL (vol. pitched into wort)
•    B. glossy (CMY-002) 26.40mL/2=13.20mL (vol. pitched into wort)

Tertiary pitching rate = 1.5×10^6cells/mL×12°P×1800mL=3.24×1010cells
•    B. claussenii (WY5151) 20.00mL×1.5=30.00mL (vol. pitched into wort)
•    B. lambicus (WY5526) 15.5mL×1.5=23.25mL (vol. pitched into wort)
•    B. matted (CMY-001) 36.61×1.5=54.91mL (vol. pitched into wort)
•    B. glossy (CMY-002) 26.40×1.5=39.60mL (vol. pitched into wort)

Second Tier: Varying Lactic Acid Concentrations
•    Trial fermentations conducted in duplicate in 1000mL Duran glass bottles. The dimensions were 100mm:230mm (diameter:height) or a ratio of 1:2.3 with a conical headspace of 20% volume.
•    Anaerobic fermentations were conducted by inserting rubber bungs air tight into the tops of the glass bottles and connecting rubber air tubing, allowing blow-off of Carbon dioxide gasses into sterile water.
•    .9L (900mL) of standard (autoclaved) wort used in each cylinder with 10ppm dissolved oxygen. (roughly 1ppm per °Plato)
•    Fermentations conducted at 21-22°C (70-72°F) in temperature-controlled incubators.
•    Pitching rate described previously (1.0×106cells/mL×12°P×900mL=1.08×1010cells)
•    When counting cells for determining amounts to be weighed out and pitched the same methods were used as previously described.
•    4 different concentrations of Lactic acid (100ppm, 500ppm, 1000ppm, 3000ppm) added to wort (after autoclaving, before aerating/pitching yeast)
•    Lactic Acid (VWR) (89.70% wt/vol, D/L%)
•    Calculation used to achieve desired concentrations:
•    100ppm: .1mL/L×.900mL×(100/89.7)=0.1003mL of Lactic acid
•    500ppm: .5mL/L×.900mL×(100/89.7)=.501mL of Lactic acid
•    1000ppm: 1.0mL/L×.900mL×(100/89.7)=1.003mL of Lactic acid
•    3000ppm: 3.0mL/L×.900mL×(100/89.7)=3.01mL of Lactic acid

100 ppm
•    B. claussenii (WLP645) Starting pH= 4.55
•    B. claussenii (WY5151) Starting pH=4.55
•    B. bruxellensis (WLP650) Starting pH=4.55
•    B. bruxellensis (WY5112) Starting pH=4.55
•    B. lambicus (WLP653) Starting pH=4.55
•    B. lambicus (WY5526) Starting pH=4.55
•    B. matted (CMY-001) Starting pH=4.55
•    B. glossy (CMY-002) Starting pH=4.55

500 ppm
•    B. claussenii (WLP645) Starting pH=4.05
•    B. claussenii (WY5151) Starting pH=4.05
•    B. bruxellensis (WLP650) Starting pH=4.05
•    B. bruxellensis (WY5112) Starting pH=4.05
•    B. lambicus (WLP653) Starting pH=4.05
•    B. lambicus (WY5526) Starting pH=4.05
•    B. matted (CMY-001) Starting pH=4.05
•    B. glossy (CMY-002) Starting pH=4.05

1000 ppm
•    B. claussenii (WLP645) Starting pH=3.75
•    B. claussenii (WY5151) Starting pH=3.75
•    B. bruxellensis (WLP650) Starting pH=3.75
•    B. bruxellensis (WY5112) Starting pH=3.75
•    B. lambicus (WLP653) Starting pH=3.75
•    B. lambicus (WY5526) Starting pH=3.75
•    B. matted (CMY-001) Starting pH=3.75
•    B. glossy (CMY-002) Starting pH=3.75

3000 ppm
•    B. claussenii (WLP645) Starting pH=3.08
•    B. claussenii (WY5151) Starting pH=3.08
•    B. bruxellensis (WLP650) Starting pH=3.08
•    B. bruxellensis (WY5112) Starting pH=3.08
•    B. lambicus (WLP653) Starting pH=3.08
•    B. lambicus (WY5526) Starting pH=3.08
•    B. matted (CMY-001) Starting pH=3.08
•    B. glossy (CMY-002) Starting pH=3.08

Using MYPG as the Growth Substrate

While most of the propagations I conducted in the lab used the same wort as was used for the Fermentation trials, I also looked at how propagations proceeded when a liquid substrate of MYPG was used. Using the three strains from Wyeast, acquired on slants, 100mL of MYPG substrate was inoculated with a loop full of cells.

As you can see there is little to no difference in cell growth based on the substrate after one day. Only B. lambicus grown in wort has slightly higher cell counts while B. bruxellensis grown in wort was not detected.

The following graph shows the growth that occurred in each 100mL of substrate from Day 1 to Day 6. The dashed lines represent the strains grown in MYPG while the black lines represent the strains grown in the standard wort substrate.  Viability was calculated and taken into account in the graph below, showing only the cell count of viable cells.

It is easy to see that the strains grown in MYPG substrate had faster growth with larger cell counts. Only B. claussenii had higher a cell count when grown in standard wort substrate.

After 6 days of growth in the MYPG substrate or Wort substrate all the flasks were taken and added to 500mL of wort substrate. Further the growth of the strains in wort substrate was observed to see if the initial substrates made a difference in the continued cell growth.

As can be seen from the graph those strains which initially were grown in MYPG substrate had higher cell counts and grew with a linear growth rate. I had previously never seen growth this rapid before with such high cell counts. It would appear that MYPG is a better suited substrate for initial culturing of cells and when looking to propagate up cells for pitching into fermentations.

Propagation Data and Graphs

7/27/09 – Monday
I have compiled all the data I received from the propagations I conducted with Brettanomyces spp. yeasts. The following three graphs contain data from the standardized propagation method I explained in the previous post using 12°Plato (1.048 gravity) wort as the growth medium. The graphs should be pretty easy to follow so I will leave it up to you to interpret. Feel free to comment on anything you find interesting in the graphs, maybe a trend you’re seeing within a strain or anything else…

This was the total cells per mL, not taking into account viability of the cells.

This is the total cells per mL which are viable.

This was the daily % of cells which appeared viable after using the methylene blue staining technique.

*Methylene Blue staining technique… The use of methylene blue to stain cells in order to determine viability proved difficult with Brettanomyces spp. I found often that cells which were viable would take up the methylene blue into the vacuoles, giving a false positive. Some would take it into the cell cytoplasm but not into the vacuole. The dye would only appear near the tips giving the cells two blue dots at each end. I have spoke with other brewers who have had trouble using methylene blue to yield positive results when doing viability checks on Brettanomyces strains. I would say from the lab experience I had and daily cell counts that an alternative method would be advantageous as I don’t believe methylene blue yields accurate results, at best it’s results which are difficult to put any sort of faith in.

Propagations Standardized

7/8/09 – Wednesday
Its been a long time since the last update. Part of the reason is I’m no longer living in Edinburgh, Scotland as I have moved to Ft. Collins, Colorado and taken a full time position with Odell Brewing Co. as a brewer. I’m also working in the cellar with the fermentations, filtering and some assistant lab work. So much for open source and updating as things finish up in the lab.
I’m working to get all the research data put up here and to continue updating as analysis of the fermentations come in. Further, now that I’m working as a professional brewer I will continue The Brettanomyces Project as an extension project and will update on experiments within the brewery. This will include filling barrels with various strains of Brettanomyces for secondary fermentations, production of American Wild Ales, cleaning and sanitation involved within a brewery when using wild yeasts, and hopefully some 100% Brettanomyces spp. primary fermentations available at the brewery.

April/May
During month of April and the beginning of May, I worked to standardize my propagations for use in the fermentations. I finished observing colony morphology and growth characteristics on various mediums and chose MYPG and WLN as my two mediums of choice. With 8 strains of Brettanomyces to work with and needing to do each propagation in duplicate to replicate the experiments it meant using multiple orbital incubators. The following is the conditions which I found to produce the highest concentration of cells the fastest, and with a great viability.
-Started with single colonies from MYPG agar, inoculated into 100mL of wort in a 250mL flask.
-All flask were first autoclaved with a sterile foam bung and aluminum foil loosely around the top.
-Initial propagation from single colonies was allowed to go for 7 days.
-The initial propagations were then decanted into 500mL of wort in a 1L flask.
-This second propagation step went 7 days before it was used for pitching into the fermentations.
-All propagations occurred in orbital incubators rotating at 80 rpm.
-Temperature set 28°C in the incubator.
-Semi aerobic conditions due to foam bung in the conical flask tops with aluminum foil over.

*A previous post or two mentions the propagations as they were going and some variations in the length of the initial propagation from the colonies, in the end the info above is what I found to work the best and within a reasonable amount of time. A video is also posted showing a set of propagations.

Wyeast to the Rescue

4/30/09 – Thursday
Over the past month or so I have been e-mailing with Greg Doss of Wyeast and it turns out the gracious people over at Wyeast were able to supply the Brettanomyces Masters Project with three slants. B. bruxellensis, B lambicus, and B. claussenii were shipped over from the USA and have arrived safely and highly viable. This brings the strain count up to 8 different strains to use in the research!
The slopes were looped and inoculated into 6 sets of conical flasks containing 100ml of solution for propagating up to pitchable counts. I went with 3 MYPG solution filled flasks and 3 autoclaved wort agar flasks to do a quick observation test of the difference in growth. Conditions are the same as with all propagations: temperatures of 28°C in th incubator with agitation around 80rpm, semi aerobic conditions with foam bung in the conical flask tops with aluminum foil over.

5/6/09 – Wednesday
What I found was interesting. I found non-cultureable cells to be highest in all three of the MYPG solution flasks. While after 6 days the non-cultureables from wort solution was at nearly zero for each of the 3 strains.
As for cell growth a different pattern was observed. For B. bruxellensis the wort/mypg cell ratio average per square was 228.4/104, for B. lambicus 125.2/76.8 and for B. claussenii 117/134.2. This shows that the yeast have different growth behaviors based on the composition of the media. This could be important in choosing the right wort to use for a 100% Brettanomyces fermentation, or when propagating up for pitching into a primary or secondary fermentation.
The flasks were then added to 500ml of sterilized wort solution. As they are grown up into pitchable amounts I will take daily cells counts monitoring the cell growth to see if the initial media continues to have an effect or not.
All propagation data from the 8 strains will be put into graphs and made available as soon as I get the last of the data.

 

Wyeast 5112 B. bruxellensis

 

 

 

 

 

Wyeast 5526 B. lambicus

 

 

 

 

 

 

Wyeast 5151 B. claussenii

A-15 Discoveries and Announcements

4/23/09 – Thursday
As can be seen when reading through the blog, I have been working with a Brettanomyces strain acquired from culturing out of an Avery 15 Anniversary ale. They refer to the strain as Drie Fonteinen, due to its original origin being cultured from a Drie Fonteinen gueuze bottle by the Brewing-Science Institute. I have been re-culturing the cells from the Avery 15 bottle over and over again for the past 3 months yet reported very little in the way of findings, just some photos here and there. Well I have made a repeated discovery and am finally confident enough (process repeated 4 times) to say that the Drie Fonteinen strain is actually culturing up as two different and quite unique Brettanomyces strains. When looking at the photos listed in the post below, there is a matted phenotype and a glossy phenotype on MYPG agar. On WLN the two form different shaped colonies and the colors are slightly different. On CuSO4 agar shown only once below the two colonies differ in the matted forms intense brain like structure with a beige color while the glossy as a round domed glossy white colonies. Under the microscope there is also a slight difference but one which is noticeable. The matted colonies are more rugby ball shaped and slightly larger then the jelly bean/cylindrical tiny glossy phenotype cells. Both strains are nearly half the size of the strains which White Labs offers. In the future I plan to do PCR to identify the the species of Brettanomyces, but for now they are being propagated up with daily cells counts being taken to observe growth phases for propagation and then for use into primary fermentations.

Going in another direction, over the past couple of weeks I have been in talking with Greg Doss the QC manager at Wyeast and they are helping out the Brettanomyces Masters Project by shipping over slopes of B. lambicus (5526), B. bruxellensis (5112), and the VSS for April to June B. claussenii (5151). Thanks to Greg for his help so far and I look forward to receiving the cultures.

Last is the newest part to the research. As the project currently has 5 pure culture, single strain species of Brettanomyces it is time to start observing propagation growth phases. All 5 strains were each inoculated into 100mL of wort solution from the standard wort produced to be used throughout the entire project. The loop contained cells from only a single colony on the respective plates and was inoculated into the wort solution. These 5 strains then propagated up over a 16 day period. At that time they were pitched into 500mL of the new sterile wort agar. From here I will be taking daily cell counts to observe the growth phases for each strain during propagation in order to observe when is best to pitch for fermentations and get a better idea of the ideal propagation to be used with Brettanomyces species. Watch for the results in the future and the possibility of further experiments which look to further maximize cell growth during propagation.

The Many Faces of Brettanomyces

4/20/09 – Monday
When I undertook this project I never imagined the microbiology involved and the hours I would spend in the lab just to get this project off the ground. Three months in the lab culturing organisms so far and I have found that just working with Brettanomyces spp. on various plate culture media can prove difficult, and takes very careful examination to ensure pure cultures are kept and maintained. Most of the past couple of months have involved working with the Brettanomyces species used by Avery Brewing Co. in their 15th Anniversary ale and cultures from yeast companies. I have cultured the Brettanomyces strains onto various medias listed previously and observed the morphology and growth habits of each strain. The following is details of how 5 Brettanomyces strains grow on the medias and pictures of their various and unique morphology. Along with culturing the different strains on various medias, careful observation under the microscope helps to observe strains general cell morphology but in no way gives a good indication to whether a pure culture is being observed due to the many growth phases and various cell shapes observed by a single culture. I will have photos later and details about cell morphology under a microscope as that is too detailed to include in this post. For now here is the photos of Brettanomyces spp. cultured on various petri plates, and is useful for any one trying to culture this incredibly fickle organism. The most difficult part of observing Brettanomyces is that little is written about the behavior and life cycle, if much is even known at all. I have discovered that even when a single colony is taken and streaked onto the agar plates not every resulting colony will grow exactly uniform or have exactly the same color or size. This could be due to stage of the life cycle when streaked from a single colony or another phenomena, but it seems to be observed in nearly every strain and is unique to Brettanomyces spp.

 

Brettanomyces A-15 (matted). This is one of two strains which makes up the “Drie Fonteinen strain” Avery uses for their pure Brettanomyces beers. It has a matted appearance on MYPG.

 

 

 

 

B. A-15 (matted). Brilliant morphology observed on WLN agar. The yeast forms a crown like effect on the edge with a nipple in the center. This is the same species as in the above photo.

 

 

 

 

B. A-15 (matted). Again brilliant morphology observed on CuSO4 agar. An extreme brain looking formation. Same species as the above two.

 

 

 

 

 

Brettanomyces A-15 (glossy). This is the second of two species which make up the “Drie Fonteinen strain” used by Avery. MYPG agar. The morphology is clearly different from the matted strain.

 

 

 

 

B. A-15 (glossy) on WLN agar. Again different from the matted strain on WLN agar.

 

 

 

 

 

Brettanomyces bruxellensis (WLP650) on MYPG agar. Nearly all the colonies for a sand dollar like pattern on top of the culture with a round dome in the middle. Very distinct and unique.

 

 

 

 

B. bruxellensis (WLP650) on WLN agar. Nearly all the colonies for a brain like pattern on top of the culture. Very distinct and unique.

 

 

 

 

Brettanomyces claussenii (WLP645) grown on MYPG agar. White normal round colonies with a rather flat look.

 

 

 

 

 

B. claussenii (WLP645) grown on MYPG+cycloheximide agar. A distinctive beige color is observable with the cycloheximide.

 

 

 

 

 

B. claussenii (WLP645) grown on WLN at 4 days since streaking. This species breaks the rules and does not metabolize the Bromocresol Green indicator as all literature states. First discovery, look how green the colonies remain.

 

 

 

 

B. claussenii (WLP645) grown on WLN at 7 days since streaking. Colonies still enlarging.

 

 

 

 

 

 

B. claussenii (WLP645) grown on WLN at 10 days since streaking. Noticeable white dots appear on the top of the colony.

 

 

 

 

Brettanomyces lambicus (WLP653) colonies which display a unique curved ring like top structure. This is on MYPG agar.

 

 

 

 

A single B. lambicus (WLP653) colony grown on WLN media with the ring like top growth on top of the colony. The other giant colonies are Saccharomyes sp.

 

 

 

 

A single B. lambicus (WLP653) colony on WLN media growing into the side of a Saccharomyces species from a mixed culture. I have found Brettanomyces species like to grow on top of and within other yeasts.

Media Cultureablity of Brettanomyces species

4/6/09 – Monday
This past week I went back and inoculated pure cultures of Brettanomyces spp. onto various agar media to observe their ability to grow on each medium. The following table is the data I received. All mediums were tested in duplicate to ensure accurate results. Incubation temperature was at 28°C for 7 days.

	B. claussenii	B. bruxellensis	B. lambicus	A-15 matted	A-15 glossy
Lysine agar	+	+	+	-	-
WLN agar	+	+	+	+	+
MYPG agar	+	+	+	+	+
MYPG+CaCO3 agar	+	+	+	+	+

Day in the Pilot Brewery

3/18/09 – Wednesday
Finally everything has progressed to the point where I can produce the standard 12°Plato (1.048) wort which will be used for every mini-fermentation throughout the Brettanomyces Masters Project. The grain bill was simple, consisting of 100% lager malt supplied by Crisps Maltings UK. The wort was hopped to 22 IBU’s using German Hallertauer Magnum hops for a 60 minute boil.

University Pilot Brewery, with a Muera 2001 mashfilter

Once the whirlpooling commenced, the wort was cooled through a heat exchanger and collected into 2 litre sterile containers for freezing for continue use through out all the experiments. The following is the explanation of the first tier of experiments.

Fun with White Labs

3/13/09 – Friday
Finally got a chance to plate out the 3 White Labs vials which came in last week. As I need to make back ups of all the Brettanomyces spp. which I collect in order to continuously re-propagate throughout the research, I made up 6 petri plates for each strain. The medium used was MYPG agar as this is my go to medium for storage and growth throughout the entire research project. The idea was 3 plates inoculated with .5mL of suspended yeast cells and 3 plates inoculated with .1mL of suspended yeast cells. After pipetting the suspended yeast cells they were dispersed using glass beads. Sometime was also allowed for the .5mL to soak into the medium since it is a large amount solution for the plates. The idea behind these inoculations was to get heavy uniform growth on the plates in order to have large amounts of growth for propagating up into flasks for future fermentations. After inoculations were done plates were put into the growth chamber at 28°C to colonize.

 

Pipetting out the B. claussenii
(WLP 645)

 

 

 

 

 

Pipetting done and 1L starter
in Erlenmeyer flask labeled
and inoculated

 

 

 

 

 

The 3 White Labs
Bretts in the Orbital
Incubator for propagation

 

 

 

The rest of the suspended yeast cells left after the pipette inoculations was poured into 1L starters. The 1.5L Erlenmeyer flasks were made up with 1L of MYPG solution and autoclaved as stated in agar recipes posted earlier. The flasks were placed into the Gallenkamp – Orbital Incubator with constant shaking at 80 rpm, and a constant temperature of 28°C.

WLN agar medium

Wallerstein Laboratory Nutrient (WLN) is a medium which is prepared already and sold by various lab companies. I used WLN from the Oxiod company with an additional of 1% (1 g/L) agar to help further solidify the agar for better streaking ability. Here is a link to the properties of WLN agar medium. The Bromocresol Green acts as a dye which Saccharomyces yeast take up and don’t normally metabolize (strain dependent). The agar medium which starts off green becomes clear/opaque as the Bromocresol Green is taken up by the developing yeast colonies. Brettanomyces spp. will also take up the Bromocresol Green but appear to metabolize it leaving Brettanomyces sp. colonies an off white to yellowish color. This medium is therefore good as a differentiation medium for observing mixed yeast cultures.
*Some warning: Not all Saccharomyces spp. take up the Bromocresol Green the same way so there are different shades of green and it is reported that some Saccharomyces spp. can also metabolize the Bromocresol Green so care must be taken if this medium is to be used for identification and it should be one of a few tests done in differentiating. Also it is possible that Brettanmyces spp. only metabolize varying amounts of the Bromocresol Green so astute observations should be made when trying to decide if only a single strain is present on a plate and plating should be done out in serial dilutions so cell counts are low enough to allow ample growth of all possible organisms present.

 

WLN agar with green medium
and Brettanomyces sp. colonies
just starting to form

 

 

 

 

After the Brettanomyces sp.
colonies have grown and all
the Bromocresol Green is
taken up

 

 

 

 

Plate inoculated with Brettanomyces sp. at
a serial dilution of 10^-6

 

 

 

 

Mixed Brettanomyces spp.
colonies? Observed color change
after a few days in the fridge,
possible strain dependent
phenomenon of not fully
metabolizing the Br. Green

 

 

 

Mixed Saccharomyces spp.
M-strain distillers yeast used
to inoculate plate

New Brett Strains

3/4/09 – Wednesday
Over the past week, one of the lab technicians tried revived an old (1978) freeze dried culture originally obtained from the NCYC. It was simply labeled as Brettanomyces claussenii (N2). This is now listed as Dekkera anomala (NCYC# 2). *UPDATE – The old freeze dried cultures were not Brettanomyces spp. and I never ended up acquiring any cultures from NCYC.

3/5/09 – Thursday
White Labs arrives! B. claussenii (WLP645) B. bruxellensis (WPL650) and B. lambicus (WLP653) have all arrived and will be placed on slopes for storage, along with propagating up to be pitched at various cell count rates. The first part of my research will involve observing pitching rates and it’s effects on the fermentations.

One Brett So Many Plates

2/23/09 – Monday
I knew I had made up a lot of plates but when it comes to critically observing each plate for growth and deciding how I should follow up with the results on the plates, it can be daunting. It is counter balanced when observing the aroma of the plates though, absolutely amazing!

The Lysine agar is worthless for culture growth. Maybe it works well with bacterias but Brettanomyces is not a fan. I had read some strains can sustain growth on Lysine and some can’t so figured it was worth observing. I found the other medias to have much better results.

This streak had been culturing for 5 days, no colonies, only minute growth.

The following MYPG+cycloheximide worked brilliantly, they had great uniform growth. The streaks gave pure colonies and looks to be a great selective media.

Streak plate with good uniform growth and colonies

10^-4 dilution

10^-6 dilution – Some of the colonies while uniform in shape and texture appear to have a brownish tinge. This may be due to being acclimated to beer and showing a slight difference in morphological color.

My absolute favorite plates were the Copper Sulphate agar plates. The aroma from these was amazing. Tropical juicy fruit Hawaiian punch, some with a slight cocoa buttery aroma. If this is any indication of the aromas produced during fermentation this strain is a winner.
A solid streak with uniform observable growth and morphology. Good pure colonies.

10^-2 dilution – These culture smells amazing. The best aromas come from the highest cell concentrated plates. Something to keep in mind when deciding what pitching rates to observe for optimal fermentations.
10^-6 dilution – Smaller colonies then the other plates appeared on the CuSO4 agar.

From the CuSO4 agar 10^-6 dilution plates and the MYPG+cycloheximide 10^-6 dilution plates single colony tops where taken with an inoculating loop and streaked onto MYPG agar. (I was careful not to take the whole colony and only the top portion. This assures I have pure cells for streaking with, in the even that I actually had a mixed yeast culture.) The same technique was used to take a colony from each of the streak plates of CuSO4 agar and MYPG+cycloheximide agar.

I’ve deciding to further observe Brettanomyces morphology on another agar medium. I prepared WLN agar plates (Oxiod) which I can use with any of the various streak plates or dilution plates. This is done by looping cells (even pipetting dregs) and inoculating into distilled water, then pipetting onto the WLN agar. I can further use this medium to observe that Brettanomyces is the only species present during the fermentations as Saccharomyces spp. will form green colonies and take up the green indicator die present in the medium, while the Brettanomyces will be observed as white/opaque colonies.

Choosing Selective Mediums

2/16/09 – Monday lab prep
Made up 3 different mediums to promote the selective growth of Brettanomyces sp. and use for enumeration. I will test these out now and save them for further use throughout the study to confirm my fermentations are uniquely Brettanomyces spp. They are MYPG with an addition of 10ppm cycloheximide (actidione), Copper Sulphate agar, and Lysine agar. These three mediums should inhibit the growth of any Saccharomyces yeasts which could be hiding, and allow me to obtain a pure colony usable throughput the rest of the study. The ingredients for making up the plates are listed below.
MYPG+cycloheximide(10ppm) agar – 500ml
Malt extract 1.5g
Yeast extract 1.5g
Peptone 1.0g
Glucose 5g
Agar 7.5g
Cycloheximide 5mg (10ppm=10mg/L in .5L=5mg)
*pH adjusted to 5.0

Copper Sulphate agar – 500ml
Malt extract Agar 50.0g
Copper Sulphate 0.6g
*Autoclaved at 115°C for 10 minutes

Lysine agar – 500ml
Glucose (1%) – 5g
L-Lysine (1%) – 5g
Agar #1 (1.2%) – 6g
Yeast salts 1mL
*pH adjusted to 6.0
*Autoclaved at 121°C for 15 minutes at 15 psi
*Add vitamin solution once cooled to pouring temp – 1mL

As always the plates were left to dry and I will streak and pipette inoculate tomorrow.
*For anyone interested in reading on selective culturing of any brewing bugs “Brewing Microbiology” by F.G. Priest has the information on page# 378-382. I highly recommend having a look through this book for all aspects on the microbiology involved in brewing.

2/17/09 – Tuesday Inoculation day
From each of the four flasks used to propagate the Brettanomyces sp. I inoculated the three different mediums (MYPG+cycloheximide agar, Copper Sulphate agar, Lysine agar). First technique was to streak inoculate. Dipping the loop into the first flask I streaked onto a CuSO4 plate then flamed and repeated with the next flask, till all four flasks had been streak inoculated. I then repeated this for the other 2 mediums. Finally the same was done with serial dilutions I made from the four flasks which were 10^-2, 10^-4 and 10^-6. Again repeating with each medium type I pipetted .1ml onto each plate and then dispersed with glass beads.
I placed them into the 28°C growth chamber and will check on their progress and the results throughout the week.

2/18/09 – Thursday Observation
Only the streaked plates had visible growth but no cell colonies to streak with. The pipetted plates had small cellular growth about to occur but no positive results yet.
I will wait till next Monday when growth should be sufficient enough to obtain single colonies to re-streak with and enumerate the cells on the plates.

Propagation Observation

2/10/09 – Tuesday propagation observation
There is no visible growth in the Brettanomyces propagation flasks yet. All flasks contain clear medium still.
There is no growth on the MYPG agar plates from the single colonies streaks. I will give it 48 hours and check back again on both.

2/12/09 – Thursday propagation observation
All flasks have visible cell growth. The flasks are cloudy compared to the clear flasks which were observed on Tuesday. Flasks believed to have Brettanomyces have some funk and slight sour acetic aroma.
All streak plates have visible growth and an acetic acid type aroma with slight funk. Some ‘bread-like’ aroma also noticed.

Day 3

*Next step is on Monday to prepare plates to further confirm the pure culture of Brettanomyces by streaking with cells from the propagated flasks onto Saccharomyces growth resistant medium

A-15 Culture Plates

2/6/09 – Friday observations
I observed the plates for the first time after a 48 hour incubation period. There was some barely observable growth, a colony or two, on 2 of the MYPG plates and 1 of the MYPG w/CaCO3. No observable smells yet other then the plate media. I left the plates to further incubate over the weekend.

2/9/09 – Monday observations
Growth has bloomed on all the plates. (Photos attached below show the extent) There is an obvious Brettanomyces funk smell to most of the plates. I found the plates containing CaCO3 to be an unnecessary culture medium. There were fewer colony forming units (CFU’s), combined with slower overall growth on every CaCO3 plate compared with the MYPG plates. This could be a coincidence but I no longer feel it advantageous to use this medium and will discard the remaining plates. For further plating of cultures I will only be using MYPG agar plates.

MYPG w/out CaCO3

This steak inoculation was done by submersing the loop in the dregs
This plate was inoculated with .1mL of pipetted dregs

This plate was inoculatedwith 1mL of pipetted dregs

MYPG w/CaCO3

This plate was inoculated with .1mL of pipetted dregs

This plate was inoculated with .1mL of pipetted dregs

This plate was inoculated with 1mL of pipetted dregs

2/9/09 – Further
4 isolated colonies from the various petri dishes were taken with a sterile loop and transfered into 100mL YPD solution in 250mL Erlenmeyer flasks for propagation. They were plugged with a sponge like porous material in the neck and aluminum foil covering the top. These will be grown up at 28°C with constant shaking at 100 rpm in a Gallenkamp – Orbital Incubator. I will check back every 24 hours to observe the propagation.

Inoculation (A-15 dregs)

2/4/09 – First Inoculation
A few days previous to inoculating, I removed the special foil and any glue used to hold it, from the top and underside of the Avery 15 bottle cap, by thoroughly cleaning and scrubbing in warm soapy water. After it was soaked in sanitizing solution and the bottle was sat up right to let the yeast cells re-collect in the bottom.
*This was done upon a recommendation by Matt “Handtruck” Thrall QA/QC for Avery Brewing.

From the plates poured a day earlier, I pulled out 6 MYPG w/CaCO3 and 6 SMYPG w/out CaCO3. Using sterile lab methods throughout the entire following procedures I opened the bottle flamed the top with a lit Bunsen burner and poured the beer into a 1,000mL glass with screw lid which I then closed. I left approximately 90mL in the bottom (too much maybe) and thoroughly roused the dregs giving a good mixing. The dregs were then poured into 5, 20mL pre-sterilized vials with screw tops.

-(6 total) 3 of each agar plate were inoculated with .1mL of dregs pipetted onto the plate then dispersed via glass beads
-(4 total) 2 of each agar plate were inoculated via loop streaking
-(2 total) 1 of each agar plate had 1mL pipetted onto the plate then dispersed via glass beads

The plates were then placed upside down labeled accordingly and placed in a culturing chamber set at 25° Celsius.

I will leave them for an initial 48 hours and keep observing till cell growth has occurred and I’m able to collect single colonies for species identification and propagation. I will post photos of the plates to show the growth and any possible zones of clearing on the CaCO3 inoculated plates if acids are produced that quickly.

Lab Preparations

2/3/09 – Day 1 in the Lab
Made up two additions of 500mL, modified MYPG agar. One with regular Malt extract and an addition of CaCO3 and the other with DME (spray malt) and no CaCO3. The calcium carbonate is supposed to help with long term storage and neutralizing the effects of acids produced by the Brettanomyces yeasts. Another ingredient which could be included is Thiamine a water-soluble vitamin of the B-complex. The phosphate derivatives are involved in many cellular processes. The best characterized form is thiamine diphosphate coenzyme used in the catabolism of sugars and amino acids. In yeast, thiamine diphosphate is required in the first step of alcoholic fermentation..
The ingredients list is:

MYPG w/CaCO3 – 500ml
-Malt extract 1.5g
-Yeast extract(mycological) 1.5g
-Peptone 1g
-Glucose 5.0g
-CaCO3 (2%) 10g
-Agar 7.5g
adjusted to pH=5.0

MYPG w/out CaCO3 – 500ml
-Malt extract 1.5g
-Yeast extract(mycological) 1.5g
-Peptone 1g
-Glucose 5g
-Agar 7.5g
adjusted to pH=5.0

Autoclaved at a rate of 15 minutes per litre. After letting them sit in a cool water bath I prepared the petri dishes. From these two solutions I poured 21 petri dishes w/out CaCO3 and 23 petri dishes w/CaCO3. I will need to work on my pouring skills… The solution with CaCO3 dropped a precipitate very quickly upon cooling and required shaking to keep in solution. The plates were turned up side down and stored to dry for 24 hours before inoculation with the dregs of Avery 15, a 100% Brettanomyces fermented beer.

-UPDATE 2/9/09
I found the plates containing CaCO3 to be an unnecessary culture medium. There were fewer colony forming units (CFU’s), combined with slower overall growth on every CaCO3 plates compared with the SMYPG plates. This could be a coincidence but I no longer feel it advantageous to use this medium. For further plating of cultures I will only be using MYPG agar plates.

The Beginning of something WILD

The Brettanomyces Masters Project:
This MSc. dissertation came about through my constant interest in Sour/Wild ales and their unique flavor. I started in October 2008, researching all the scientific papers I could find that were written regarding Brettanomyces spp. I wrote up a preliminary literature review further delving into the microbiological and biochemical side of this wild yeast. Throughout my search for scientific literature and studies, I was able to find limited amounts about this yeast through various brewing, wine and industrial alcohol studies. I found that little to no literature existed on Brettanomyces spp. as primary fermentation yeast in alcohol beverages and all knowledge of this yeast with concern to brewing is from either Lambic or Flemish Red production research.
All the while a few creative craft brewers in the US have created such a beer and an even larger proportions of homebrewers are constantly creating innovative beers using only Brettanomyces spp. Most importantly is a forum known as the Burgundian Babble Belt which is dominated by Brettanomyces topics and possibly the most knowledgeable individuals when it comes to brewing with only Brettanomyces spp.
So how does one use Brettnomyces spp. as a primary fermentation yeast, under what fermentation conditions is an optimal beer produced, what compounds are produced in what quantities, and what about anaerobic vs. aerobic environment and the so called Custers effect, ect… with those questions I introduce my research.

MSc. Dissertation – My research involves observing the characteristics of various Brettanomyces spp. (B. bruxellensis, B. lambicus, and B. anomalus). In order to gain an idea of how these yeasts can be used in the brewhouse I’ll be focusing on the following areas of research:
-Culturability onto various mediums and observing colony morphology.
-Standardizing propagation methods and observing cell biomass production.
-Conducting multiple primary fermentations in order to find optimal conditions for an anaerobic beer fermentation.

Further, I’m particularly interested in the compounds produced during the fermentations due to the various conditions. Studies have been done on the production of esters, specifically ethyl acetate, ethyl lactate, ethyl caproate, ethyl caprylate, ethyl caprate, 4-ethyl phenol, 4-ethyl guiaicol, tetrahydropyridines, and lowered levels of isoamyl acetate by Brettanomyces spp. But these were always looked at with regards to Lambic production, Sour ale production, or as a secondary fermentation in wine. I’m concerned with observing the amounts of compounds produced during a 100% Brettanomyces primary fermentation. Where are the various fruity aromas coming from, is it really ethyl lactate? Through adding varying amounts of lactic acid to the fermentations I can observe the formation of ethyl lactate and continue where other research has left off. Compounds produced will be quantified through Headspace GC, HPLC, and further analysis in an effort to correlate the production of compounds with the specific fermentation conditions. Along with this I will be analyzing various other primary and secondary fermented beer to observe differences the wort and starting conditions make.
I have attached my lit review “The Horse the Goat and the Barnyard” with a few alterations and highlights of the take away message. The review paper makes for a chunky, yet informative background read of the historical uses and research which has been carried out on Brettanomyces spp. so far…
*Due to a 5,000 word limit set on the paper (which I maxed by another 2,500 words) the review does not contain information on acetic acid production by Brettonamyces spp. due to aerobic conditions, observations of anaerobic culturing or the limiting growth factors of anaerobic conditions. I’m looking to get this updated as soon as possible and turn it into more of a complete overview of Brettanomyces spp. and the research done to date.

I would ask anyone interested to subscribe to my blog if you want to follow this open source Masters dissertation from start to finish on Brettanomyces spp. and their use as a primary fermentation yeast.

-Chad Y.