Research Methodology for Pure Culture Fermentation

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.

~ by Chad on October 8, 2009.

5 Responses to “Research Methodology for Pure Culture Fermentation”

  1. Chad,
    By Anaerobic fermentation do you mean that the wort received no aeration whatsoever, or do you mean that it was not exposed to oxygen after fermentation had started?

    • Hey Sean,

      All the fermentation started with a dissolved oxygen level between 10ppm-12ppm, obtained by diffusing oxygen into the wort and measuring with an ORBISPHERE 3650, O2 analyzer. Afterward the fermentations proceeded in an anaerobic state as would occur in standard fermentations conducted in cylindro-conical fermenters. This is different then say fermentations conducted in a carboy with just a wod dowel in the top, or a barrel where diffusion of oxygen occurs into the beer so it is not a strictly anaerobic environment.


  2. Chad,

    I’m wondering if you’ve looked into using straight lactose in B.Anomalous studies of ethyl lactate. B. brux and B. Lamb are reported to not ferment lactose. I’ve done a few experiements and found that the B. Ano does well with lactose and the lactose doesn’t monkey up the pH of the wort. Worked out well with both mixed ferments (reg yeast + brett) and singular ferment with B ano. This actually works extremely well in sweet stout which is were the B. ano was discovered anyway (stout that is).

    See The yeasts: a taxonomic study By C. P. Kurtzman, Jack W. Fell for a deeper info.

    Kristen England, Ph.D.
    BJCP Education Director

    • Hi Kristen,

      I have not done any studies using lactose as a fermentable compound which would produce lactic acid and ethyl alcohol, both nicely acidifying the wort and adding alcohol while giving both the precursors for ethyl lactate to be formed. I wanted to do test tube trials with the yeasts I have to observe which strains would metabolize which saccharides and carbohydrates, but have not gotten around to it. In order to further the Brettanomyces research I would need to enter into a Ph.D program as there is so much to be explored at the cellular level and through conducting a range of further Primary/Secondary fermentations.
      As far as I know, I don’t have the B. anomalus strain, this is the second time this week some one has asked me about that strain. I tried to get it from NCYC but ended up starting the project as I already had 8 strains. 6 of which appear to be B. bruxellensis after PCR with the exception that I’ve not looked into doing PCR for the two B. claussenii strains yet, they should both be B. anomalus but yeast labs are quirky so no telling what they are….
      This is a really interesting idea and another way of approaching the topic. I would be interested in talking this over more with you through email. As for the elusive book, The yeasts: a taxonomic study… I’ve been trying to get a hold of this book since I started my research but have not found it in any University Libraries I have access too, and am unable to fork out the $460… I really need that book…


  3. Chad,

    Anytime, you have my email and we can discuss in length at your leisure.


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