Moving Homes

•December 1, 2010 • Leave a Comment

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

Chad Y.

Dissertation Submitted

•July 23, 2010 • 3 Comments

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.

Brettanomyces Yeast Cell Images

•June 30, 2010 • 1 Comment

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
WLP650 B. bruxellensis

Allagash house Brettanomyces
Allagash’s house Brettanomyces strain

Girardin Gueuze 1 waxy-white
strain cultured
from Girardin Gueuze

Girardin Gueuze 2 glossy-white
2nd Brettanomyces strain cultured
from Girardin Gueuze

Girardin Gueuze 3 glossy-white-tan
3rd Brettanomyces strain cultured
from Girardin Gueuze

WY 5112 B. bruxellensis
WY5112 B. Bruxellensis

Data Collection Methods of Analysis

•December 3, 2009 • Comments Off on Data Collection Methods of Analysis

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:

  1. Final Gravity
  2. Final pH

Compound Analysis:

  1. Headspace Gas Chromatography – FID
    Ethyl Acetate
    Ethyl Lactate
    Isobutyl Acetate
    Ethyl Butyrate
    Isoamyl Acetate
    Ethyl Caproate
    Ethyl Caprylate
  2. Headspace Gas Chromatography – ECD
  3. High Performance Liquid Chromatography
  4. High Performance Anion Exchange with Pulsed Amperometric Detector (0.5ml×2) – Determination of sugars present (HPAE-PAD)

Brettanomyces Research Fermentation Photos

•November 1, 2009 • Comments Off on Brettanomyces Research Fermentation Photos

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.

WLP/BSI/CMY primary fermentationsCMY001 & BSI-Drie strains along with
WhiteLabs strains in a temperature
controlled incubator

Close up of Fermentations in incubatorClose up with some of the fermentations
initially containing lactic acid

WY/WLP/BSI/CMY pitchrate primary fermentationsCMY001 & BSI-Drie, Wyeast, and WhiteLabs
strains with different pitch rates in a
temperature controlled incubator

Wyeast Primary FermentationPreparing Wyeast strains before moving to a
temperature controlled environment

WLP650 surface pellicleSurface yeast or pellicle forming on
WLP650 B. bruxellensis

Close up of surface pellicleClose up of the thin crusty pellicle

Research Methodology for Pure Culture Fermentation

•October 8, 2009 • 5 Comments

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.

Species Identification

•October 1, 2009 • Comments Off on Species Identification

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).