The Brettanomyces Project

Moving Homes

Chad — Wed, 01 Dec 2010 19:12:35 +0000

Since finishing writing for the dissertation, I’ve been working hard to get the entire contents of the dissertation posted as well as moving all the past posts to a new website. The new website is now up and operational with the dissertation content posted and a new section up which will detail the practical usage of Brettanomyces yeasts during my upcoming endeavor. Check out the new website and additional content.

the Brettanomyces Project

Cheers,
Chad Y.

Filed under: Brettanomyces Project

Dissertation Submitted

Chad — Fri, 23 Jul 2010 22:10:53 +0000

I’m excited to say I finally finished up writing and have submitted my dissertation on Brettanomyces yeasts titled “Pure Culture Fermentation Characteristics of Brettanomyces Yeast Species and Their Use in the Brewing Industry”. The project took an additional five months of compiling data and writing to get it all finished. In the end, I’m very proud of all that I have learned and plan to keep sharing the funk while expanding the knowledge of this yeast genus. The entire contents of the dissertation is available and can be reached by visiting the The Brettanomyces Masters Dissertation.

Thanks to everyone who followed the blog during my studies and make sure to check out all the information I compiled throughout the study. Additionally I plan to see how many unique beers I can conceive through starting a brewery of my own, look forward to more information as it becomes available.

-Chad Y.

Filed under: MSc. Project

Brettanomyces Yeast Cell Images

Chad — Wed, 30 Jun 2010 20:01:19 +0000

While lately I’ve mostly been concentrating on writing and compiling all the information necessary to complete this dissertation, I recently took some time to present a lot of my findings to the Fort Collins Home brew club The Liquid Poets. During this I was able to meet Remi, the yeast wrangler for the home brew club. Remi and I have been comparing strains which we’ve cultured from various sources and as of this I have been able to acquire a few new strains for my ever growing Brettanomyces collection. Remi has also been nice enough to share his photos of some of the Brettanomyces yeast cells taken at 400 X magnififcation. Thanks to Remi for sharing his yeast cell photos.

WLP650 B. bruxellensis

Allagash’s house Brettanomyces strain

Brettanomyces strain cultured
from Girardin Gueuze

2nd Brettanomyces strain cultured
from Girardin Gueuze

3rd Brettanomyces strain cultured
from Girardin Gueuze

WY5112 B. Bruxellensis

Filed under: MSc. Project, Yeast cell images

Data Collection Methods of Analysis

Chad — Thu, 03 Dec 2009 21:05:04 +0000

Pure culture fermentations were given 35 days (5 weeks) to attenuate before data and samples were collected and frozen for later analysis. Samples of un-fermented wort had previously been collected and frozen for comparative analysis along with samples from the batch culture propagations. It may be useful to observe the different quantities of compounds produced during semi-aerobic batch culture propagation as compared to compounds that are produced during anaerobic fermentation. Below details the data collected after pure culture fermentation along with the compounds which were observed. When collecting biological samples for compound analysis all volumes were collected in double to allow duplicate runs, eliminating the window of error during a run. The following link details the Methods of Analysis used during the various fermentation trials as it was written in the dissertation.

Data collected:

Final Gravity
Final pH

Compound Analysis:

Headspace Gas Chromatography – FID
Acetaldehyde
Ethyl Acetate
Ethyl Lactate
Isobutyl Acetate
Ethyl Butyrate
Isoamyl Acetate
Ethyl Caproate
Ethyl Caprylate
n-Propanol
Isobutanol
2-Methylbutanol
3-Methylbutanol
Headspace Gas Chromatography – ECD
Diacetyl
2,3-Pentanedione
High Performance Liquid Chromatography
4-Vinylphenol
4-Vinylguaiacol
High Performance Anion Exchange with Pulsed Amperometric Detector (0.5ml×2) – Determination of sugars present (HPAE-PAD)
Glucose
Fructose
Sucrose
Maltose
Maltotriose

Posted in MSc. Project, Project overview

Brettanomyces Research Fermentation Photos

Chad — Mon, 02 Nov 2009 04:34:57 +0000

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

CMY001 & BSI-Drie strains along with
WhiteLabs strains in a temperature
controlled incubator

Close up with some of the fermentations
initially containing lactic acid

CMY001 & BSI-Drie, 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 crusty pellicle

Posted in Fermentation, MSc. Project, Project overview

Research Methodology for Pure Culture Fermentation

Chad — Thu, 08 Oct 2009 18:06:56 +0000

While performing the propagation trials, I prepared the research methodology for the pure culture fermentations that would make up the bulk of the research. As previously mentioned all fermentations were conducted with wort produced on the 2 hectolitre pilot system. The following details the trials I put together for researching the characteristics of primary fermentation with eight Brettanomyces strains. The purpose of the pure culture fermentation research is to also observe what compounds are produced during fermentation, and how those concentrations compare to the levels commonly associated with beer. Most recently, tropical fruit descriptors have become sought after aromas having anecdotally been reported from fermentations using pure cultures of Brettanomyces spp. It is not known which strains create the pineapple-like ester characteristics, although it has become the goal of a handful of brewers to recreate techniques that have resulted in these light fruity attributes. A handful of theories exist for the possible sources of the unique aromas with no study previously concentrating on a wide variety of secondary metabolites produced by Brettanomyces yeasts. In doing so, two variables were observed during pure culture anaerobic fermentation; pitching rate and initial lactic acid concentration. Three different pitching rates were studied: 6×10^6, 12×10^6, and 18×10^6 cells/ml and five initial lactic acid concentrations: 0, 100, 500, 1,000, and 3,000 mg/l in order to evaluate their influence on the over all fermentation performance. The information that can be provided from this study regarding strains available from commercial yeast companies allows conclusions to be made about the best use of each strain and methods which might maximize their ability to produce various aromatic compounds including the occasionally described pineapple and tropical fruit characteristics.

Impact of Pitching Rate

Trial fermentations were conducted in duplicate in 2,000 ml Duran glass bottles
Fermenter vessel dimensions were 136 mm:265 mm (diameter:height) or a ratio of 1:1.96 with a conical headspace of 20% volume
1,800 ml of sterilized wort was used with ~10 ppm dissolved oxygen, diffused into the wort prior to pitching the yeast (roughly 1 ppm DO per °Plato)
Anaerobic fermentation was conducted by inserting rubber bungs air tight into the tops of the glass bottles and connecting rubber air tubing through the center, allowing blow-off of Carbon dioxide gas into sterile water
Fermentation was 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
When counting cells with a hemocytometer for determining amounts to be measured out for pitching, viability was also determined and recorded, then taken into account to attain 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.

Primary pitching rate = 12×10^6 cells/ml
Each of the eight Brettanomyces strains were inoculated into pure culture fermentations using the industry standard pitching rate for Saccharomyces ale strains (1×10^6 cells/ml/°P). This was chosen as the primary pitching with two other pitching rates chosen for comparisons to be made.

Secondary pitching rate = 6×10^6 cells/ml
Tertiary pitching rate = 18×10^6 cells/ml
The secondary and tertiary pitching rates involved observing four of the Brettanomyces strains at half the primary pitching rate and one and a half times the primary pitching rate. Based on the fermentation performance at each pitching rate a comparative analysis can be done observing the effect of the multiple pitching rates.

Impact of Varying Lactic Acid Concentrations

Trial fermentations were conducted in duplicate in 1,000 ml Duran glass bottles
The dimensions were 101 mm:230 mm (diameter:height) or a ratio of 1:2.27 with a conical headspace of 20% volume
Anaerobic fermentation was conducted by inserting rubber bungs air tight into the tops of the glass bottles and connecting rubber air tubing through the center, allowing blow-off of Carbon dioxide gas into sterile water
900 ml of sterilized wort was used with ~10 ppm dissolved oxygen, diffused into the wort prior to pitching the yeast (roughly 1 ppm DO per °Plato)
Fermentation was conducted at 21-22°C (70-72°F) in temperature-controlled incubators
Primary pitching rate (12×10^6 cells/ml) for fermentation
When counting cells for determining amounts to be measured out and pitched, the same methods were used as previously described
Four different concentrations of lactic acid (100, 500, 1,000, 3,000 mg/l) added to wort (after autoclaving, before aerating/pitching yeast)
Lactic Acid (VWR) (89.70% wt/vol)

Low pH environments are the conditions which Brettanomyces yeasts are most commonly associated with, yet no previous studies have been conducted to observe the effects of low pH on the combined fermentation performance by this yeast genus. For this study, wort was acidified with lactic acid before pitching the yeast and comparative analysis will made concerning each fermentation. The following shows the pH at the start of fermentation after the various lactic acid additions.

Control 0 mg/l
Starting pH=4.95
100 mg/l
Starting pH= 4.55
500 mg/l
Starting pH=4.05
1,000 mg/l
Starting pH=3.75
3,000 mg/l
Starting pH=3.08

Check out the following link to read more about the Pure Culture Fermentation methods and results from the dissertation content posted on-line.

Posted in Fermentation, MSc. Project, Project overview

Species Identification

Chad — Thu, 01 Oct 2009 22:02:57 +0000

Over the past couple of months I’ve been emailing with a graduate student at the University of California, Santa Cruz who was interested in running PCR identification on the two Brettanomyces strains (CMY001 and BSI-Drie) along with a few others. I just received an email detailing that PCR identification sequencing had been carried out on three of the Brettanomyces spp. used in this study and another that I have since acquired. It was determined that both the CMY001 and BSI-Drie strains are Brettanomyces bruxellensis and Wyeast 5151 is Brettanomyces anomalus. Thanks goes out to Michael Pearson for carrying out the PCR identification sequencing. The method used for identification was based on polymorphism in the rRNA Internal Transcribed Spacer Region. To read more about identifying Brettanomyces yeasts I’ve posted the methods used for the Identification of the “Drie” strain(s).

Posted in MSc. Project

MYPG Compared to Wort as a Growth Substrate

Chad — Sat, 08 Aug 2009 19:21:37 +0000

While most of the batch culture propagations were conducted using wort as the growth substrate, I also looked at how batch culture growth proceeded using MYPG as the growth substrate. This study was performed with each of the three Brettanomyces strains from Wyeast by inoculating them into 100 ml of MYPG substrate and 100 ml of wort substrate. The substrates were inoculated by looping cells from a single colony grown on MYPG agar. Initial cell growth in both mediums proceeded for 7 days before the propagations were poured aseptically into 500 ml of wort in order to observe any difference the starting medium had over subsequent growth. The second propagation step underwent a further 7 days of cell growth with comparisons made regarding overall cell growth.

Growth curve of three Wyeast strains initially sub-cultured in MYPG substrate
as compared to the same strains initially cultured separately in wort substrate.
Both substrates were further inoculated into 500ml of wort substrate and
grown for 168 hours (7 days) at 28°C with 80-rpm agitation.

Growth curve comparison between the collective means of three Wyeast strains
initially sub-cultured in MYPG substrate as compared to the same strains
initially cultured separately in wort substrate. Both substrates were further
inoculated into 500ml of wort substrate and grown for 168 hours (7 days)
to observe initial impact on cell growth. Mean cell count data was obtained
from all three Wyeast strains grown at 28°C with 80-rpm agitation.

I had previously not had growth this rapid before yielding 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 fermentation. More information on Propagation and Batch Culture Growth can be found by following the link.

Posted in Cell growth/biomass, MSc. Project, Propagation

Propagation Data and Graphs

Chad — Tue, 28 Jul 2009 04:10:49 +0000

I have finished compiling the data on batch culture propagations conducted with five of the Brettanomyces strains. The following two graphs contain data from the standardized propagation method described in the previous post using 12 °Plato (1.048 gravity) wort as the growth medium. Some variation between growth patterns can be seen along with what appears to be step-like growth until the stationary phase is reached after a period of 168 to 192 hours. More information on Propagation and Batch Culture Growth can be found by following the link.

Growth curve for five strains of Brettanomyces during semi-aerobic batch culture.
Cultures were grown in 500 ml of wort substrate over a 288-hour
period at 28°C with 80-rpm agitation. Viability was taken into
account to reflect the actual cell number.

Daily percentage of cells which appeared viable after using the
methylene blue staining technique. Cultures were grown in
500ml of wort substrate over a 288-hour period at 28°C
with 80-rpm agitation.

*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 completely accurate results.

Posted in Cell growth/biomass, MSc. Project, Propagation

Propagations Standardized

Chad — Fri, 15 May 2009 21:40:00 +0000

During the month of April and beginning of May, I worked to standardize batch culture propagation methods for maximizing cell growth. I had previously finished observing colony morphology and growth characteristics on various mediums and chose MYPG and WLN agar as my two mediums of choice. With eight strains of Brettanomyces to use throughout the research and needing to propagate each strain in duplicate (in order to replicate the experiments) I had to use multiple orbital incubators and tied up a lot of the University lab’s resources. The following is the conditions which I found to produce the highest concentration of cells which were physically ready to start fermentation with high vitality and viability.

Started with single colonies from MYPG agar, inoculated into 100 ml of wort in a 250 ml flask.
All flask pre-filled with the wort solution were 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 aseptically into an additional 500 ml of wort in a 1.5 liter flask.
This second propagation step went 8 days before it was used for pitching into fermentations.
All propagations occurred in orbital incubators rotating at 80 rpm.
Temperature was set at 28°C in the incubator.
Semi-aerobic conditions were present as a result of using a foam bung in the conical flask tops with aluminum foil covering.

A previous post or two mentions the propagation methods 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 used throughout the rest of the study to propagate yeast cells. Check out the following link to read more about the Propagation and Batch Culture Methods used throughout the dissertation.

Posted in MSc. Project, Propagation

Wyeast to the Rescue

Chad — Wed, 06 May 2009 15:04:00 +0000

4/30/09 – Thursday

Over the past month or so I have been e-mailing with Greg Doss of Wyeast Labs as previously mentioned and it turns out the gracious people over at Wyeast were able to supply the Brettanomyces Masters Project with three slopes. WY5112 (B. bruxellensis), WY5526 (B. lambicus) and WY5151 (B. claussenii) were shipped over from the USA and have arrived safely. This brings the strain count up to eight different strains that are being used in the research!

The slopes were looped and inoculated into 6 sets of 250 ml conical flasks containing 100 ml of solution for propagating up to pitchable amounts. I will be observing the growth during batch culture in two different mediums this time. Of the six flasks the strains were inoculated into, three contain MYPG solution and three contain wort solution. This is a quick observation to test the difference in growth between the two mediums. Conditions are the same as with all the batch culture propagations: Temperatures of 28°C in the Orbital incubator with agitation around 80 rpm, semi aerobic conditions with foam bung in the conical flask tops and aluminum foil covering.

5/6/09 – Wednesday

What I observed was interesting. I found a slightly higher amount of non-viable cells in all three of the MYPG solution flasks as the over all viability was 96.68%, while after 6 days the cells grown in wort solution had 99.73% viability overall. As for cell growth a different pattern was observed. For B. bruxellensis the wort/mypg cell ratio average per square on the hemocytometer 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 cells for pitching into a primary or secondary fermentation. The flasks were then added to 500 ml 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 eight 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

Posted in Cell growth/biomass, Culturing, MSc. Project, Propagation

Media Cultureablity of Brettanomyces Species

Chad — Fri, 01 May 2009 02:34:00 +0000

Screening of Various Media Agars

I have finished collecting and compiling data on the ability of B. bruxellensis (WLP650), B. lambicus (WLP653), B. claussenii (WLP645), B. bruxellensis (BSI-Drie) and B. bruxellensis (CMY001) to culture on popular brewery related media agar. This is the first part of the research and was carried out to find the relevance of each media, its use throughout the project, and practicability of use in the brewing industry. The observations should be useful for QA/QC labs wanting to culture and maintaining various Brettanomyces strains. The strength of colony growth was judged based on the time it took for single colonies to develop with distinguishable morphological traits and the ease with which each medium could be used to enumerate and identify strains. The medias which colonies were observed on were MYPG, MYPGcc, MYPGch, WLN, WLD, CuSO₄, Lysine and UBA agars. To read more about the Evaluation of Yeast Culturability on various media agar check out the link or you can find more information on strain specific Morphological Traits on various media agars.

Media	WLP645	WLP650	WLP653	CMY001	BSI-Drie
MYPG	++	++	++	++	++
MYPGcc	nd	+	+	+	+
MYPGch	++	++	++	++	++
WLN	++	++	++	++	++
WLD	++	++	++	++	++
Lysine	+/-	+/-	+/-	-	-
CuSO4	++	++	++	++	++
UBA	+	+	+/-	+/-	+

-, No detectable growth; +/-, weak growth; +, moderate growth; ++, strong growth; nd, not determined.

Posted in Culturing, MSc. Project

The Many Faces of Brettanomyces

Chad — Tue, 21 Apr 2009 00:43:00 +0000

Giant Colony Morphologies
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 media agars can prove difficult. Each plate takes careful examination to ensure pure cultures are kept and maintained. Overtime this becomes easier and I have started to memorize traits each strain possess on the different media agars. The majority of the past few months have involved working with the Brettanomyces species cultured from Avery’s 15th Anniversary ale and cultures from White Labs yeast company. I have been culturing the Brettanomyces strains onto various medias listed previously and observed the morphologies and growth habits of each strain. The following is photos from the three White Labs Brettanomyces strains and their unique morphologies on various media agars. 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 at one time. I will try to post photos later and detail cell morphology under a microscope but that is too detailed to include in this post. For now here are some photos of the Brettanomyces spp. cultured on the various agar plates as this may be 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 a stage of the life cycle when streaked from a single colony or another phenomena, but it seems to be observed in nearly every strain.

Brettanomyces bruxellensis (WLP650) on MYPG agar
Nearly all the colonies form a sand dollar
like pattern on top of the culture
with a round dome in the middle

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

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 the literature states
New 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) on MYPG agar
colonies display a unique curved
ring like top structure

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

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

Posted in Colony morphology photos, Culturing, MSc. Project, Project overview

Project Updates

Chad — Mon, 13 Apr 2009 02:38:00 +0000

Over the past couple of weeks I have been in talking with Greg Doss the QC manager at Wyeast labs and they will be 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 and I look forward to receiving the cultures once they are shipped and clear customs!

As the project continues, I now have five pure culture strains of Brettanomyces and have started observing cell growth phases during batch culture propagation. All five strains were seperatly inoculated into 250 ml flasks containing 100 ml of wort solution taken from the standard wort produced and being used throughout the entire project. The flasks containing the wort were loop inoculated with cells from the top of a single colony grown on MYPG agar plates. These five strains were then propagated up over a 16 day period and at that time the 100 ml yeast slurry was pitched into 500 ml of new sterile wort solution and grown in a 1.5 litre flask. From here I will again 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 fermentation and get a better idea of the ideal propagation methods to be used with Brettanomyces yeast species. Watch for the results in the future and the possibility of further experiments which look to further maximize cell growth during propagation.

Posted in MSc. Project, Project overview

Two for One Brettanomyces Strains

Chad — Mon, 06 Apr 2009 17:30:57 +0000

The “Drie” strain(s)
As can be seen from reading through the blog, I have been repeatedly culturing what I thought was a single Brettanomyces strain acquired from the dregs of a bottle of Avery’s 15th Anniversary ale. The strain they used for the 100% Brettanomyces fermentation is referred to as “Drie“, as it was 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 2 months yet have reported very little in the way of findings, just some photos here and there. What I have repeatedly observed and am finally confident enough (process repeated 4 times) to say is that the “Drie” strain has actually cultured up as two different and quite unique Brettanomyces strains. When looking at the photos listed below, there is a matted phenotype and a glossy phenotype on MYPG agar. On WLN agar the two strains form different shaped colonies and the colors are slightly different also. On CuSO4 agar the two colonies differ as the matted phenotype forms an intense brain like domed structure with a beige color while the glossy phenotype develops a volcano shaped beige colored round glossy colonies with an off-white center tip. Under the microscope there is also a slight difference in the cells, one which is noticeable. The matted phenotype cells are 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 two species of Brettanomyces, but for now they are being propagated up separately with daily cells counts being taken to observe growth phases during batch culture propagation and then for use in pure culture fermentation.
I don’t know the exact reason why there is two morphologically different strains culturing up when only one was used to ferment the beer. My guess is that over time the original strain has mutated possibly due to environmental conditions and a second strain has become established. I might possibly be able to tell more after looking at fermentation characteristics, but for now the glossy phenotype is believed to be the “Drie” strain and the matted phenotype is a completely unique new strain.

CMY001 (matted phenotype) on MYPG agar

CMY001 (matted phenotype) on WLN agar
This yeast forms a crown like effect
on the edge with a nipple
in the center

CMY001 (matted phenotype) on CuSO4 agar
An extreme brain looking morphology

BSI-Drie (glossy phenotype) on MYPG agar

BSI-Drie (glossy phentoype) on WLN agar.
Younger colonies starting to take shape

Posted in Bottle culturing, Colony morphology photos, MSc. Project, Project overview

Day in the Pilot Brewery

Chad — Wed, 18 Mar 2009 21:14:00 +0000

Wednesday Pilot Brewing
Finally everything has progressed to the point where I can produce a standard 12°Plato (1.048) wort which will be used for each batch propagation and mini-fermentation throughout the Brettanomyces Masters Project. The ICBD pilot brewery is a custom built brewery which produces 2 hectoliter (200 litre) batch volumes. The grain bill for the brew was simple consisting of 100% lager malt supplied by Crisps Maltings, UK. Instead of brewing with the often standard British mash-tun and conducting a single infusion mash we produced the wort using a mash filter from Muera. The mash filter is slightly different as the malt is ground finely with a hammer mill and mashed into a mash mixer for conversion of enzymes. The mash is then pumped over to the mash filter where strong worts are first extracted and then water from the hot liquor tank is sparged through the filter plates/screens for collection of the remaining sugars which run-off into the kettle. The wort was hopped to 22 IBU’s using German Hallertauer Magnum hops with a 60 minute boil.

University Pilot Brewery, with the Muera 2001 Mash filter

After boiling the wort and whirlpooling had commenced, the wort was cooled through the heat exchanger and collected into about 100, 2 litre sterile containers to freeze for continual use throughout the project.

Posted in Brewing, MSc. Project

Fun with White Labs

Chad — Fri, 13 Mar 2009 22:04:00 +0000

Friday Lab Happenings
Finally got a chance to plate out the three 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 possibly re-propagate throughout the research, I inoculated 2 MYPG agar plates for each strain. The plates were inoculated with .1 ml of suspended yeast slurry and dispersed using glass beads. After inoculating, the plates were placed in the growth chamber at 28°C. Single colonies will then be transferred from the plates onto multiple MYPG agar slopes. MYPG agar has become my go to medium for storage and growth throughout the entire research project.

Pipetting out B. claussenii
(WLP 645)

Done pipetting with a 1 litre starter
labeled and inoculated

The three White Labs
Brett strains propagating
in the Orbital incubator

The remaining yeast suspensions were each poured into separate 1 litre starters. The 1.5 litre Erlenmeyer flasks were made up with 1 litre of MYPG solution without the addition of agar and autoclaved as stated in the MYPG recipes posted earlier. The flasks were then placed into the Gallenkamp – Orbital Incubator with constant shaking at 80 rpm and a temperature of 28°C.

Posted in Culturing, MSc. Project, Propagation

WLN Agar Medium

Chad — Sat, 07 Mar 2009 00:18:00 +0000

A New Selective Media Agar
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 1% (1 g/l) of agar added to help further solidify the media for better streaking ability without gouging into the media. Here is a link to the properties of WLN agar medium. The Bromocresol Green present in the medium 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 take up the Bromocresol Green but have been observed to metabolize the dye leaving the Brettanomyces yeast colonies an often cyan, or yellowish off white 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. (Wit ale strains!) 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. (B. claussenii!) 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.

Wallerstein Laboratory Nutrient agar – 1,000 ml
WLN 75 g
Agar (additional 1%) 10 g
*Autoclaved at 121°C for 15 minutes at 15 psi

Wallerstein Laboratory Nutrient+cycloheximide (WLD) agar – 1,000 ml
WLN 75 g
Agar (additional 1%) 10 g
*Autoclaved at 121°C for 15 minutes at 15 psi
*Add Cycloheximide after autoclaving media – 10 mg (10 ppm=10 mg/l)

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

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

Posted in Agar media recipes, Culturing, MSc. Project

White Labs Brettanomyces Strains

Chad — Fri, 06 Mar 2009 00:21:00 +0000

White Labs yeasts arrive! Brettanomyces claussenii (WLP645), Brettanomyces bruxellensis (WPL650) and Brettanomyces lambicus (WLP653) have all arrived and will be inoculated onto MYPG agar plates and onto MYPG agar slopes for storage. I will also be propagating up each of the strains to observe growth curves during batch culture propagation. This will be the second part of the research with the objective to find a standard propagation method used for growing up pitchable quantities of Brettanomyces yeast cells for primary fermentation.

Posted in MSc. Project, Project overview

One Brett So Many Plates

Chad — Mon, 23 Feb 2009 23:02:00 +0000

Monday Observations
I knew I had made up a lot of plates but when it came to critically observing each plate for growth and deciding how I should follow up with the results, it becomes time consuming and taking good notes is critical. It is counter balanced when observing the aroma of the plates though, some are absolutely amazing!

The Lysine agar media used in this study observed weak growth of colonies. 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 five days, no colonies, only stunted growth.

Weak growth after five days

The following MYPG+cycloheximide agar media worked brilliantly, the plates had great uniform growth with the streaks forming solid single colonies. This media looks to be a great selective media.

This streak plate had good uniform growth
and single colonies already forming

10^-4 serial dilution

10^-6 serial dilution – Some
of the colonies while uniform
in shape and texture appear
to have a brownish tinge

My absolute favorite plates were the Copper Sulfate agar media. The aroma from these was amazing. Tropical juicy fruit, Hawaiian punch, and 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 single colonies forming.

10^-2 dilution – This culture smelled 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
were observed on CuSO4 agar
then the other medias.

From the 10^-6 serial diluted CuSO4 agar plates and the 10^-6 serial diluted MYPG+cycloheximide agar plates, single colony tops where looped and streak inoculated onto MYPG agar. Care was taken to only take the top portion of the colony as this assures that pure single cells are looped for streaking in the event that non-cultureable Saccharomyces cells are laying dormant near to a Brettanomyces colony. 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 spp. morphology on another agar medium. I prepared Wallerstein Laboratory Nutrient (WLN) agar plates, which I can further use to observe that Brettanomyces is the only species present during the fermentations as Saccharomyces spp. will form green colored colonies through take up a green pH indicator dye present in the medium, while the Brettanomyces should be observed as white colored colonies.

Posted in Culturing, MSc. Project

Inoculation for Pure Culture Confirmation

Chad — Fri, 20 Feb 2009 01:37:57 +0000

2/17/09 – Tuesday Inoculation day
Using the yeast slurry from each of the four flasks used to propagate up the Avery 15 Brettanomyces sp., I inoculated three different mediums (Copper Sulfate, MYPG+cycloheximide and Lysine agar) prepared the day earlier. First technique was to loop cells from the slurry and streak inoculate each of the different mediums. Dipping the loop into the first flask I streaked onto a CuSO4 plate then flamed the loop and proceeded to inoculated from the same flask onto each of the other two media agars. I repeated these steps with the next flask until all four flasks had been streak inoculated onto each of the three mediums. The same was then done with serial dilutions I made from the four flasks. The serial dilutions were performed at 10^-2, 10^-4 and 10^-6 times the propagate yeast slurry attained from each flask. Again repeating as I’d done when streaking with each medium type, I pipetted .1 ml onto each plate and then dispersed with glass beads. When finished I placed the inoculated plates into the 28°C growth chamber and plan to check on their progress and the results throughout the week.

Copper Sulfate, MYPG+cycloheximide and Lysine agar plates

2/19/09 – Thursday Observation
Only the streaked plates had visible growth but no single colonies had formed yet. The pipetted plates had small cellular growth about the size of a pin head. Checking the colonies after 48 hours is too soon and four to five days seems to be a better wait time before first checking for colony growth. I will wait till next Monday (2/23/09) when growth should be sufficient enough to obtain single colonies to re-streak with and enumerate the colonies on the plates.

Posted in Culturing, MSc. Project

Choosing Selective Agar Medias

Chad — Mon, 16 Feb 2009 22:20:00 +0000

Monday Lab Prep
Made up three different media agars to promote the selective growth of Brettanomyces spp. and use for enumeration. I will test these out with the currently propagating cultures and save them for further use throughout the study to confirm my fermentations are uniquely Brettanomyces spp. The three media agars I chose to prepare were MYPG with an addition of 10 ppm cycloheximide (actidione), Copper Sulfate (CuSO4) and Lysine agar. These three mediums should inhibit the growth of any Saccharomyces yeasts which could be present, and allow me to obtain a pure culture usable throughout the rest of the study. The ingredients for making up the plates are listed below.

MYPG+cycloheximide(10 ppm) agar – 500 ml
Malt extract 1.5 g
Yeast extract 1.5 g
Peptone 1.0 g
Glucose 5 g
Agar 7.5 g
Cycloheximide 5 mg (10 ppm=10 mg/l in .5 l=5 mg)
*pH adjusted to 5.0

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

Lysine agar – 500 ml
Glucose (1%) 5 g
L-Lysine (1%) 5 g
Agar (1.2%) 6 g
Yeast salts 0.5 ml
*pH adjusted to 6.0
*Autoclaved at 121°C for 15 minutes at 15 psi
*Add vitamin solution once cooled to pouring temp – 1 ml/l

As always the plates were dried in the plate pouring room and I will be ready to streak and pipette inoculate some of them 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.

Posted in Agar media recipes, MSc. Project

Propagation Observation

Chad — Thu, 12 Feb 2009 21:41:00 +0000

2/10/09 – Tuesday Propagation Observation
There was no visible growth in the Brettanomyces propagation flasks yet as each flasks media solution was still unclouded. Further, there was no growth on the MYPG agar plates created from the single colony streaks. I will give it 48 hours and check back again on both.

2/12/09 – Thursday Propagation Observation
All the flasks now have visible cell growth. The flasks are cloudy compared to the flasks which were observed on Tuesday. Some of the flasks already have a slight Brettanomyces funk with a slight sour acetic aroma. All the streak plates also have visible cell growth and an acetic acid type aroma with a slight funk. Some ‘bread-like’ aroma was also noticed.

Day 3 of Propagation

The next step is on Monday (2/16/09) to prepare a few new agar medias, before streaking the propagating flasks out onto different Saccharomyces growth resistant mediums and MYPG agar to confirm these are pure cultures of a Brettanomyces sp.

Posted in Culturing, MSc. Project, Propagation

Avery 15 Culture Plates

Chad — Mon, 09 Feb 2009 18:23:00 +0000

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 the MYPG plates and one of the MYPG w/CaCO3 plates. No observable smells yet other then the agar media. I left the plates to further incubate over the weekend.

2/9/09 – Monday Observations
Growth has bloomed on all the plates in five days. There is an obvious Brettanomyces funk smell to most of the plates. I’ve 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 standard MYPG plates. This could be a coincidence but I no longer feel it advantageous to use this medium. For further plating of cultures I will be using MYPG agar plates. The plates will be left in the incubator to further observe giant morphologies.

MYPG agar
This steak inoculation was done by submersing the loop in the dregs

This plate was inoculated with .1 ml of pipetted dregs

This plate was inoculated with 1 ml of pipetted dregs

MYPG w/CaCO3

This plate was inoculated with .1 ml of pipetted dregs

After observing the plates I isolated 4 colonies from the various petri dishes. The single colonies were looped and transferred into 100 ml of YPD solution in a 250 ml Erlenmeyer flasks for propagation. A foam bung was inserted into the neck of the flask and the top was covered with a square piece of aluminum foil. These will be grown up at 28°C with constant shaking at 100 rpm in a Gallenkamp – Orbital Incubator. The same four colonies were also looped and streaked onto MYPG agar before being placed into the culturing chamber at 28°C. I will check back every 24 hours to observe the cell growth.

Posted in Culturing, MSc. Project

Inoculating and Culturing the Dregs of Avery 15

Chad — Wed, 04 Feb 2009 07:13:00 +0000

Culturing Yeast From Avery’s 15th Anniversary Beer
A few days prior to inoculating plates, I removed the special foil (and glue used to hold the foil) from the top and underside of an Avery 15 bottle cap by thoroughly cleaning and scrubbing with warm soapy water. After rinsing and drying the bottle, the neck of the bottle including the cap was soaked in sanitizing solution and the bottle was sat up right to let the yeast cells re-collect in the bottom. Avery’s 15th Anniversary beer is one of the first 100% Brettanomyces fermented beers and was produced with a unique strain not available to homebrewers. The strain they used is refereed to as “Drie“, as it was cultured from a Drie Fonteinen gueuze bottle by the Brewing-Science Institute.

Using the plates poured a day earlier, I pulled out 6 MYPG w/CaCO3 and 6 standard MYPG plates. Using sterile lab techniques throughout the entire process, I opened the bottle, flamed the lip with a lit Bunsen burner and poured the beer into a glass. I left approximately 90 ml of dregs in the bottle (too much only needed about 20 ml) and thoroughly roused the dregs giving a good mixing. The dregs were then aseptically poured into 5, 20 mL pre-sterilized (autoclaved) vials with screw tops.

- 3 of each agar plate (6 total) were inoculated with .1 ml of dregs pipetted onto the plate then dispersed via glass beads
- 2 of each agar plate (4 total) were inoculated by looping cell and streaking for single colonies
- 1 of each agar plate (2 total) had 1 ml pipetted onto the plate then dispersed via glass beads (This is too much inoculate, .1 ml is more then enough!)

After the inoculate absorbed into the plates the plates were then placed upside down, labeled accordingly, and placed in a culturing chamber set at 28° 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 MYPG w/CaCO3 inoculated plates if acids are produced that quickly.

Posted in Bottle culturing, Culturing, MSc. Project

Lab Preparations

Chad — Wed, 04 Feb 2009 04:23:00 +0000

Day 1 in the Lab
Made up two additions of MYPG agar in 500 ml volumes. One was with the addition of CaCO3 and the other without CaCO3. MYPG w/CaCO3 is similar to Dekkera media agar recommended for use in culturing Brettanomyces spp. by the European Brewery Convention (EBC). 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 and the best characterized form is thiamine diphosphate a coenzyme used in the catabolism of sugars and amino acids. In yeast, thiamine diphosphate is required in the first step of alcoholic fermentation. It has been reported that Brettanomyces spp. lacks the ability to produce Thiamine and therefore exogenous additions are required for adequate growth.

Agar media ingredients list:
MYPG agar – 500 ml
-Malt extract 1.5 g
-Yeast extract 1.5 g
-Peptone 1 g
-Glucose 5 g
-Agar 7.5 g
adjusted to pH=5.0

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

Autoclaved at 121°C for 15 minutes at 15 psi. After letting the media cool in a water bath I prepared 100 mm petri dishes in a sanitary room. From these two solutions I poured 21 petri dishes of MYPG agar and 23 petri dishes of MYPG w/CaCO3 agar. 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. Once the plates had solidified they were turned up side down and angled on the lid to allow the medium 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 MYPG w/CaCO3 plates compared with the standard MYPG 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.

Posted in Agar media recipes, MSc. Project

The Beginning of something WILD

Chad — Mon, 02 Feb 2009 05:33:00 +0000

The Brettanomyces Masters Project:
This MSc. dissertation is the result of my enthusiasm and interest in Sour/Wild ales and their unique flavors and aromas. Over the past decade, Brettanomyces spp. have seen an increasing use in the craft-brewing sector of the industry with a handful of breweries having produced beers that were primary fermented with pure cultures of Brettanomyces spp. This has occurred out of experimentation as relatively little information exists regarding pure culture fermentative capabilities and the aromatic compounds produced by various strains. In October 2008, I started scouring academic databases on-line and at Heriot-Watt’s University library for all the scientific papers I could obtain regarding Brettanomyces yeasts. During the first semester of my Masters studies in Brewing and Distilling I wrote a preliminary literature review concentrating on all aspects of this yeast genus. I became predominantly interested in the microbiology and biochemistry behind pure culture fermentation, especially with regards to volatile compound production. Throughout my search for literature published in scientific journals I found numerous studies that were conducted over the past century on Brettanomyces spp., with only a limited amount of those studies relating to the brewing industry. The information available on pure culture fermentation with Brettanomyces yeasts was inconsistent and inconclusive, leaving a void in the understanding of the overall function of this yeast genus and its application as a fermentative yeast species. The majority of the recent research has focused on enhancing the knowledge of the wine industry. I intend to provide a greater knowledge of the Brettanomyces strains available in the brewing industry through focusing on strain specific fermentation characteristics and identifying the major compounds produced during pure culture anaerobic fermentation in wort.

I started by asking myself, “how does a brewer use Brettanomyces as a pure culture primary fermentation yeast? Under what fermentation conditions is an optimal beer produced? What are the typical aromatic compounds and in what quantities are they found? What about anaerobic vs. aerobic growth and/or fermentation and the so called Custers effect?” With those questions I introduce my research.

MSc. Dissertation Topics

Culturability on various agar medias and observing colony morphology
Standardizing propagation methods and observing cell biomass production
Pure culture primary fermentation:
- Observing the impact of multiple pitching rates
- Observing the impact of multiple initial lactic acid concentration in wort

I’m particularly interested in the attenuation levels of each strain and volatile, aromatic compounds produced during the fermentation due to the various conditions. Previous studies have observed the production of a few esters, specifically ethyl acetate, ethyl lactate, ethyl caproate, ethyl caprylate, ethyl caprate, and lowered levels of isoamyl acetate by Brettanomyces spp. along with the production of 4-ethyl phenol, 4-ethyl guiaicol, and tetrahydropyridines. These studies however were conducted with regards to Lambic production, Flemish Sour Red/Brown production, or during secondary fermentation in wine. It is the aim of this study to focus on observing the amounts of compounds produced during pure culture Brettanomyces primary fermentation. Through using pure cultures the various esters produced can be quantified and the source of the fruity aromas which are anecdotally reported from pure culture fermentation can be discovered? Through adding varying amounts of lactic acid to the wort prior to fermentation I can observe the formation of ethyl lactate and continue where other research has left off, while further observing the impact lowered pH has on fermentation.

I would ask anyone interested in this site and wanting to follow this open source Masters dissertation from start to finish on Brettanomyces spp. please subscribe to receive up dates.

-Chad Y.

Posted in MSc. Project, Project overview