Wild Brettanomyces Project – 2024 Update
My longtime readers will know about my Wild Brettanomyces Project – my multi-year quest to try and identify the environmental niche of Brettanomyces – e.g. the place where it lives in the wild. 2024 was no exception to this, so it’s time for a quick update. As a quick reminder, Brettanomyces are often found in large numbers in industrial beverage contexts (breweries, wineries, etc). But, oddly enough, Brettanomyces doesn’t appear to live on any of the inputs for these industries – its not on grains, or on grapes/other fruits, nor carried by insects, or found in any of the other usual locations we find other species of wild yeasts. This has made finding new strains of Brettanomyces – or avoiding it in industries where it’s an issue – difficult.
Several years ago I happened across a series of papers looking at the rhizosphere’s of various plants – e.g. at the commensal bacteria and fungi that grow around a plants roots and help the plants survive. Rhizosphere’s are not an accidental thing, and plants actively promote the growth of beneficial organisms by secreting sugars and other nutrients from their roots to feed this community. And guess what I found in several papers, often in the long lists of identified species hidden away in the supplemental figures…Brettanomyces!
Since then I have been pursuing this possibility with decidedly mixed results. While I am often able to find Brettanomyces-like cells (in terms of appearance) in rhizosphere isolates, purifying these have been challenging as another common wild yeast – Debaryomyces – readily outgrows everything else in the culture. So while I’ve had a few small successes, meaningful identification of Brettanomyces in rhizosphere samples still eludes me.
Other parts of this series:
- Part 0: Where do the Wild Brettanomyces Roam?
- Part I: These Are Not The Yeast You Are Looking For
- Part II: These ARE The Yeast Your Are Looking For
- Part III: Wild Brettanomyces – a Needle in a Haystack
- Part IV: Fermentation characteristics
- Part V: Wild Brettanomyces – The 2023 Search
- Part VI: Wild Brettanomyces – The 2024 Search (this post)
Wild Brettanomyces Project in 2024
When I last wrote about this, I was trying to optimize an enrichment medium which would allow Brettanomyces to grow while inhibiting the growth of Debaryomyces. To do this I was using a cocktail of four selective agents: 1) antibiotics to suppress bacteria. 2) alcohol to suppress moulds and other non-ethanol-tolerant organisms. 3) cycloheximide to kill cycloheximide-sensitive yeasts (which are most of them). 4) acetic acid (vinegar) to suppress Debaryomyces. I also “capped” some tubes with a layer of mineral oil to suppress oxygen-dependent growth.
While the various combinations I tested worked well at suppressing Debaryomyces while allowing some Brettanomyces growth, this only worked for pure strains. Root and other rhizosphere samples failed to produce any growth (see the picture to the left). I was unable to resolve why this was the case, but I suspect it was because I was using a brewery-adapted strain of Brettanomyces that is better at anaerobic metabolism than wild strains.
So what did I do in 2024? I tried to optimize the medium some more. It’s clear I need a combination of selective agents to suppress the growth of unwanted organisms, but it’s also clear that these selective agents are damaging to Brettanomyces. I need to find the right balance if I want this project to succeed. But this is not trivial. The antibiotics do not affect fungal growth, so they do not require optimization – but the doses of alcohol, cycloheximide, acetic acid, and oil capping – all need to be optimised. This is not a trivial task. Even if I only consider only two concentrations of each compound, that’s a total of 16 permutations! And I need to test far more than that – 72 possible combinations – based on last years work:
| Compound | Dose |
|---|---|
| Alcohol | 0%, 2%, 4% |
| Cycloheximide | 0, 50, and 100 mg/L |
| Acetic Acid | 0, 25, 50, and 100 mM |
| Oil | Used or absent |
I’ll admit that I was a little overwhelmed by this task until I came across this excellent video by Nighthawk In Light. This is a much more efficient way to perform optimization processes!
The Optimisation Process
This optimisation process uses Taguchi arrays. This is a method of performing a limited set of permutations of your variables that still allows for optimization. This is done in a way where every possible value of each variable is tested an equal amount, but not every possible combination is used. Instead, the combinations are setup to allow you to “isolate” the effect of each variable, allowing you to identify near-optimal values for each variables.
These approaches work best if you have the same number of “options” for each variable, so I simplified my test scheme by eliminating some of the options my previous tests suggest are unlikely to work. I also didn’t bother testing oil this time around. This brings the tests down to 4 from the original 16:
Table 1:
| Test | Ethanol | Cycloheximide | Acetic Acid |
|---|---|---|---|
| 1 | 2% | 50 mg/L | 25 mM |
| 2 | 2% | 100 mg/L | 50 mM |
| 3 | 4% | 50 mg/L | 50 mM |
| 4 | 4% | 100 mg/L | 25 mM |
These were then added to my standard base medium:
Table 2:
| Compound | Amount |
|---|---|
| Water | 49 mL |
| Yeast Extract | 0.5 g |
| Peptone | 1 6 |
| Dextrose (glucose) | 1 g |
| Ampicillin* | 2.5 mg |
| Chloramphenicol* | 2.5 mg |
| Selective agents* | see above table |
| * dissolved in 1 mL of sterile water |
This medium is autoclaved, then the 1 mL solution of ampicillin, chloramphenicol, and the other selective agents added.
Changes to Sampling and Analysis
I also changed my approach to sampling, based on what I read in a few papers and in discussion with some microbiology colleagues. Previously, I was growing my cultures in this medium for 14 days in the hopes of selectively growing out Brettanomyces. This wasn’t working, so instead I used this medium for negative selection – e.g. I grew the samples in it for 24 hours before plating on my conventional medium. The idea being to kill off whatever I could, and to then grow out the Brettanomyces (plus whatever else survived) on a less stressful medium. In all cases, I plated 100 uL (0.1 mL) of the medium onto my standard detection plate:
Table 3:
| Compound | Amount |
|---|---|
| Water | 200 mL |
| Yeast Extract | 2 g |
| Peptone | 4 g |
| Dextrose (glucose) | 4 g |
| Bromocresol Green | 6 mg |
| Ampicillin* | 10 mg |
| Chloramphenicol* | 10 mg |
| * dissolved in 1 mL of sterile water |
colonies on bromocresol green.
The other thing I changed was how I analyzed these samples. Because I was trying to rapidly screen for potentially useful medium I did not do a detailed (e.g. genetic) identification. Instead, I relied on a 3-part “tentative identification” process. 1) I only screened colonies that took at least 4 days to appear, 2) I only screened colonies which converted bromocresol green from a blue to clear pigment (versus an accumulation or conversion to yellow by other yeasts – see image to the left). 3) I screened the colonies that passed criteria 1 & 2 by microscopy, analyzing them for a Brettanomyces-like morphology. Slow growing, white yeast colonies, with Brett-like morphology, were assumed to be Brettanomyces for the sake of validating this approach. The goal here is quick scoring, not in-depth analysis.
Results
Table 4:
| Test | Eth/CHex/ AA* | Growth** | % White*** | Brett Morphology **** |
|---|---|---|---|---|
| 1 | 2/50/25 | High | <10% | <5% |
| 2 | 2/100/50 | Low | ~50% | ~50% |
| 3 | 4/50/50 | Medium | ~50% | ~50% |
| 4 | 4/100/25 | Low | ~25% | ~50% |
- * Ethanol (%)/Cycloheximide (mg/L)/acetic acid (mM)
- ** Relative number of non-mould colonies
- *** Portion of colonies that are white (e.g. potentially Brettanomyces)
- **** Portion of white colonies with Brettanomyces-like cells
The analysis here is somewhat interesting. Firstly, alcohol concentration doesn’t correlate highly with Brettanomyces recovery, but does reduce the number of extraneous colonies. The second is that acetic acid enriches for yeast with Brett-like morphology more than the other agents, whereas cycloheximide seems to control the total number of yeast colonies (Brett + non-Brett). What’s not reflected here is that I am still having issues with mould growth. This is a particular issue as it takes nearly a week for Brettanomyces colonies to form, during which time mould can nearly cover the plate.
Sadly, this is as far as I got in 2024…as I mentioned in my 50 meter beer project videos, last year was a crazy year outside of the brewery (silly job, getting in the way of my hobbies). But it wasn’t for not. I now have a new selective medium that I will use moving forward. I may also have found a way to address my mould issue. The cheap chemical biphenyl is a potent anti-mould agent, but does not affect the growth of the family of yeasts to which Brettanomyces belongs. So I have a plan for 2025…and hopefully the end is in sight for this project:
- I will pre-deplete the soil samples of most yeasts by culturing them for 24-48 hours in a selective medium containing 4% ethanol, 50 mM acetic acid, and 50 mg/L cycloheximide (plus the usual antibiotics to knock back the bacteria).
- The cultures from step #1 will be plated on my standard agar plate mixture (Table 3), supplemented with 100 mg/L cycloheximide and 200 mg/L biphenyl.
With luck, this will yield a good population of wild Brettanomyces for further investigation! I’ve already started preparing medium for 2025, so hopefully there will be instagram posts (or even blog posts) soon!
