{"id":3535,"date":"2025-03-21T06:55:00","date_gmt":"2025-03-21T11:55:00","guid":{"rendered":"https:\/\/suigenerisbrewing.com\/?p=3535"},"modified":"2025-03-21T06:56:01","modified_gmt":"2025-03-21T11:56:01","slug":"wild-brettanomyces-project-2024-update","status":"publish","type":"post","link":"https:\/\/suigenerisbrewing.com\/index.php\/2025\/03\/21\/wild-brettanomyces-project-2024-update\/","title":{"rendered":"Wild Brettanomyces Project &#8211; 2024 Update"},"content":{"rendered":"\n<p>My longtime readers will know about my Wild Brettanomyces Project &#8211; <a href=\"https:\/\/suigenerisbrewing.com\/index.php\/2024\/02\/10\/wild-brettanomyces-2023\/\">my multi-year quest<\/a> to try and identify the environmental niche of <em>Brettanomyces<\/em> &#8211; e.g. the place where it lives in the wild. 2024 was no exception to this, so it&#8217;s time for a quick update. As a quick reminder, <em>Brettanomyces<\/em> are often found in large numbers in industrial beverage contexts (breweries, wineries, etc). But, oddly enough, <em>Brettanomyces <\/em>doesn&#8217;t appear to live on any of the inputs for these industries &#8211; 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 <em>Brettanomyces <\/em>&#8211; or avoiding it in industries where it&#8217;s an issue &#8211; difficult.<\/p>\n\n\n\n<p>Several years ago <a href=\"https:\/\/suigenerisbrewing.com\/index.php\/2022\/07\/07\/where-do-wild-brettanomyces-live\/\">I happened across a series of papers<\/a> looking at the rhizosphere&#8217;s of various plants &#8211; e.g. at the commensal bacteria and fungi that grow around a plants roots and help the plants survive. Rhizosphere&#8217;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&#8230;<em>Brettanomyces<\/em>!<\/p>\n\n\n\n<p>Since then I have been pursuing this possibility with decidedly mixed results. While I am often able to find <em>Brettanomyces<\/em>-like cells (in terms of appearance) in rhizosphere isolates, purifying these have been challenging as another common wild yeast &#8211; <em>Debaryomyces <\/em> &#8211; readily outgrows everything else in the culture. So while I&#8217;ve <a href=\"https:\/\/suigenerisbrewing.com\/index.php\/2022\/11\/02\/this-is-the-wild-brettanomyces-your-are-looking-for\/\">had a few small successes<\/a>, meaningful identification of <em>Brettanomyces<\/em> in rhizosphere samples still eludes me.<\/p>\n\n\n\n<h4 class=\"wp-block-heading\">Other parts of this series:<\/h4>\n\n\n\n<ul class=\"wp-block-list\">\n<li>Part 0: <a href=\"https:\/\/suigenerisbrewing.com\/index.php\/2022\/07\/07\/where-do-wild-brettanomyces-live\/\">Where do the Wild Brettanomyces Roam?<\/a><\/li>\n\n\n\n<li>Part I:\u00a0<a href=\"https:\/\/suigenerisbrewing.com\/index.php\/2022\/10\/06\/this-is-not-the-yeast-your-are-looking-for\/\">These Are Not The Yeast You Are Looking For<\/a><\/li>\n\n\n\n<li>Part II:\u00a0<a href=\"https:\/\/suigenerisbrewing.com\/index.php\/2022\/11\/02\/this-is-the-wild-brettanomyces-your-are-looking-for\/\">These ARE The Yeast Your Are Looking For<\/a><\/li>\n\n\n\n<li>Part III:\u00a0<a href=\"https:\/\/suigenerisbrewing.com\/index.php\/2022\/11\/06\/wild-brettanomyces-needle\/\">Wild Brettanomyces \u2013 a Needle in a Haystack<\/a><\/li>\n\n\n\n<li>Part IV:\u00a0<a href=\"https:\/\/suigenerisbrewing.com\/index.php\/2023\/03\/24\/wild-brettanomyces-fermentation-test\/\">Fermentation characteristics<\/a><\/li>\n\n\n\n<li>Part V: <a href=\"https:\/\/suigenerisbrewing.com\/index.php\/2024\/02\/10\/wild-brettanomyces-2023\/\">Wild Brettanomyces \u2013 The 2023 Search<\/a><\/li>\n\n\n\n<li>Part VI: <a href=\"https:\/\/suigenerisbrewing.com\/index.php\/2025\/03\/21\/wild-brettanomyces-project-2024-update\/\">Wild Brettanomyces &#8211; The 2024 Search<\/a> (this post)<\/li>\n<\/ul>\n\n\n\n<hr class=\"wp-block-separator has-alpha-channel-opacity\"\/>\n\n\n\n<h2 class=\"wp-block-heading\">Wild Brettanomyces Project in 2024<\/h2>\n\n\n<div class=\"wp-block-image\">\n<figure class=\"alignleft size-medium\"><img data-recalc-dims=\"1\" loading=\"lazy\" decoding=\"async\" width=\"180\" height=\"300\" data-attachment-id=\"3360\" data-permalink=\"https:\/\/suigenerisbrewing.com\/index.php\/2024\/02\/10\/wild-brettanomyces-2023\/sec_rhizome\/\" data-orig-file=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2024\/02\/sec_rhizome.jpg?fit=689%2C1150&amp;ssl=1\" data-orig-size=\"689,1150\" data-comments-opened=\"1\" data-image-meta=\"{&quot;aperture&quot;:&quot;0&quot;,&quot;credit&quot;:&quot;&quot;,&quot;camera&quot;:&quot;&quot;,&quot;caption&quot;:&quot;&quot;,&quot;created_timestamp&quot;:&quot;0&quot;,&quot;copyright&quot;:&quot;&quot;,&quot;focal_length&quot;:&quot;0&quot;,&quot;iso&quot;:&quot;0&quot;,&quot;shutter_speed&quot;:&quot;0&quot;,&quot;title&quot;:&quot;&quot;,&quot;orientation&quot;:&quot;0&quot;}\" data-image-title=\"sec_rhizome\" data-image-description=\"\" data-image-caption=\"\" data-large-file=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2024\/02\/sec_rhizome.jpg?fit=614%2C1024&amp;ssl=1\" src=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2024\/02\/sec_rhizome.jpg?resize=180%2C300&#038;ssl=1\" alt=\"\" class=\"wp-image-3360\" srcset=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2024\/02\/sec_rhizome.jpg?resize=180%2C300&amp;ssl=1 180w, https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2024\/02\/sec_rhizome.jpg?resize=614%2C1024&amp;ssl=1 614w, https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2024\/02\/sec_rhizome.jpg?w=689&amp;ssl=1 689w\" sizes=\"auto, (max-width: 180px) 100vw, 180px\" \/><\/figure>\n<\/div>\n\n\n<p><a href=\"https:\/\/suigenerisbrewing.com\/index.php\/2024\/02\/10\/wild-brettanomyces-2023\/\">When I last wrote about this<\/a>, I was trying to optimize an enrichment medium which would allow <em>Brettanomyces <\/em>to grow while inhibiting the growth of <em>Debaryomyces<\/em>. 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 <em>Debaryomyces<\/em>. I also &#8220;capped&#8221; some tubes with a layer of mineral oil to suppress oxygen-dependent growth. <\/p>\n\n\n\n<p>While the various combinations I tested worked well at suppressing <em>Debaryomyces <\/em>while allowing some <em>Brettanomyces<\/em> 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 <em>Brettanomyces<\/em> that is better at anaerobic metabolism than wild strains.<\/p>\n\n\n\n<p>So what did I do in 2024? I tried to optimize the medium some more. It&#8217;s clear I need a combination of selective agents to suppress the growth of unwanted organisms, but it&#8217;s also clear that these selective agents are damaging to <em>Brettanomyces<\/em>. 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 &#8211; but the doses of alcohol, cycloheximide, acetic acid, and oil capping &#8211; all need to be optimised. This is not a trivial task. Even if I only consider only two concentrations of each compound, that&#8217;s a total of 16 permutations! And I need to test far more than that &#8211; 72 possible combinations &#8211; based on last years work:<\/p>\n\n\n\n<div class=\"wp-block-columns is-layout-flex wp-container-core-columns-is-layout-9d6595d7 wp-block-columns-is-layout-flex\">\n<div class=\"wp-block-column is-layout-flow wp-block-column-is-layout-flow\" style=\"flex-basis:40%\">\n<figure class=\"wp-block-table alignleft is-style-stripes has-small-font-size\"><table class=\"has-fixed-layout\"><thead><tr><th>Compound<\/th><th>Dose<\/th><\/tr><\/thead><tbody><tr><td>Alcohol<\/td><td>0%, 2%, 4%<\/td><\/tr><tr><td>Cycloheximide<\/td><td>0, 50, and 100 mg\/L<\/td><\/tr><tr><td>Acetic Acid<\/td><td>0, 25, 50, and 100 mM<\/td><\/tr><tr><td>Oil<\/td><td>Used or absent<\/td><\/tr><\/tbody><\/table><\/figure>\n<\/div>\n<\/div>\n\n\n\n<p>I&#8217;ll admit that I was a little overwhelmed by this task until I came across this excellent video by <a href=\"https:\/\/www.youtube.com\/@Nighthawkinlight\">Nighthawk In Light<\/a>. This is a much more efficient way to perform optimization processes!<\/p>\n\n\n\n<figure class=\"wp-block-embed is-type-video is-provider-youtube wp-block-embed-youtube wp-embed-aspect-16-9 wp-has-aspect-ratio\"><div class=\"wp-block-embed__wrapper\">\n<span class=\"embed-youtube\" style=\"text-align:center; display: block;\"><iframe loading=\"lazy\" class=\"youtube-player\" width=\"800\" height=\"450\" src=\"https:\/\/www.youtube.com\/embed\/5oULEuOoRd0?version=3&#038;rel=1&#038;showsearch=0&#038;showinfo=1&#038;iv_load_policy=1&#038;fs=1&#038;hl=en-US&#038;autohide=2&#038;wmode=transparent\" allowfullscreen=\"true\" style=\"border:0;\" sandbox=\"allow-scripts allow-same-origin allow-popups allow-presentation allow-popups-to-escape-sandbox\"><\/iframe><\/span>\n<\/div><\/figure>\n\n\n\n<hr class=\"wp-block-separator has-alpha-channel-opacity\"\/>\n\n\n\n<h2 class=\"wp-block-heading\">The Optimisation Process<\/h2>\n\n\n\n<p>This optimisation process uses <a href=\"https:\/\/en.wikipedia.org\/wiki\/Taguchi_methods\">Taguchi arrays<\/a>. 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 &#8220;isolate&#8221; the effect of each variable, allowing you to identify near-optimal values for each variables.<\/p>\n\n\n\n<p>These approaches work best if you have the same number of &#8220;options&#8221; 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&#8217;t bother testing oil this time around. This brings the tests down to 4 from the original 16:<\/p>\n\n\n\n<p class=\"has-small-font-size\"><strong>Table 1<\/strong>:<\/p>\n\n\n\n<div class=\"wp-block-columns is-layout-flex wp-container-core-columns-is-layout-9d6595d7 wp-block-columns-is-layout-flex\">\n<div class=\"wp-block-column is-layout-flow wp-block-column-is-layout-flow\" style=\"flex-basis:50%\">\n<figure class=\"wp-block-table is-style-stripes has-small-font-size\"><table class=\"has-fixed-layout\"><thead><tr><th class=\"has-text-align-center\" data-align=\"center\">Test<\/th><th>Ethanol<\/th><th>Cycloheximide<\/th><th>Acetic Acid<\/th><\/tr><\/thead><tbody><tr><td class=\"has-text-align-center\" data-align=\"center\">1<\/td><td>2%<\/td><td>50 mg\/L<\/td><td>25 mM<\/td><\/tr><tr><td class=\"has-text-align-center\" data-align=\"center\">2<\/td><td>2%<\/td><td>100 mg\/L<\/td><td>50 mM<\/td><\/tr><tr><td class=\"has-text-align-center\" data-align=\"center\">3<\/td><td>4%<\/td><td>50 mg\/L<\/td><td>50 mM<\/td><\/tr><tr><td class=\"has-text-align-center\" data-align=\"center\">4<\/td><td>4%<\/td><td>100 mg\/L<\/td><td>25 mM<\/td><\/tr><\/tbody><\/table><\/figure>\n<\/div>\n<\/div>\n\n\n\n<p>These were then added to my standard base medium:<\/p>\n\n\n\n<p class=\"has-small-font-size\"><strong>Table 2<\/strong>:<\/p>\n\n\n\n<div class=\"wp-block-columns is-layout-flex wp-container-core-columns-is-layout-9d6595d7 wp-block-columns-is-layout-flex\">\n<div class=\"wp-block-column is-layout-flow wp-block-column-is-layout-flow\" style=\"flex-basis:40%\">\n<figure class=\"wp-block-table is-style-stripes has-small-font-size\"><table class=\"has-fixed-layout\"><thead><tr><th>Compound<\/th><th>Amount<\/th><\/tr><\/thead><tbody><tr><td>Water<\/td><td>49 mL<\/td><\/tr><tr><td>Yeast Extract<\/td><td>0.5 g<\/td><\/tr><tr><td>Peptone<\/td><td>1 6<\/td><\/tr><tr><td>Dextrose (glucose)<\/td><td>1 g<\/td><\/tr><tr><td>Ampicillin*<\/td><td>2.5 mg<\/td><\/tr><tr><td>Chloramphenicol*<\/td><td>2.5 mg<\/td><\/tr><tr><td>Selective agents*<\/td><td>see above table<\/td><\/tr><\/tbody><tfoot><tr><td>* dissolved in 1 mL of sterile water<\/td><td><\/td><\/tr><\/tfoot><\/table><\/figure>\n<\/div>\n<\/div>\n\n\n\n<p>This medium is autoclaved, then the 1 mL solution of ampicillin, chloramphenicol, and the other selective agents added. <\/p>\n\n\n\n<hr class=\"wp-block-separator has-alpha-channel-opacity\"\/>\n\n\n\n<h2 class=\"wp-block-heading\">Changes to Sampling and Analysis<\/h2>\n\n\n\n<p>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 <em>Brettanomyces<\/em>. This wasn&#8217;t working, so instead I used this medium for negative selection &#8211; 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 <em>Brettanomyces<\/em> (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:<\/p>\n\n\n\n<p class=\"has-small-font-size\"><strong>Table 3<\/strong>:<\/p>\n\n\n\n<div class=\"wp-block-columns is-layout-flex wp-container-core-columns-is-layout-9d6595d7 wp-block-columns-is-layout-flex\">\n<div class=\"wp-block-column is-layout-flow wp-block-column-is-layout-flow\" style=\"flex-basis:40%\">\n<figure class=\"wp-block-table is-style-stripes has-small-font-size\"><table class=\"has-fixed-layout\"><thead><tr><th>Compound<\/th><th>Amount<\/th><\/tr><\/thead><tbody><tr><td>Water<\/td><td>200 mL<\/td><\/tr><tr><td>Yeast Extract<\/td><td>2 g<\/td><\/tr><tr><td>Peptone<\/td><td>4 g<\/td><\/tr><tr><td>Dextrose (glucose)<\/td><td>4 g<\/td><\/tr><tr><td>Bromocresol Green<\/td><td>6 mg<\/td><\/tr><tr><td>Ampicillin*<\/td><td>10 mg<\/td><\/tr><tr><td>Chloramphenicol*<\/td><td>10 mg<\/td><\/tr><\/tbody><tfoot><tr><td>* dissolved in 1 mL of sterile water<\/td><td><\/td><\/tr><\/tfoot><\/table><\/figure>\n<\/div>\n<\/div>\n\n\n<div class=\"wp-block-image is-style-default\">\n<figure class=\"alignleft size-medium\"><img data-recalc-dims=\"1\" loading=\"lazy\" decoding=\"async\" width=\"300\" height=\"224\" data-attachment-id=\"2940\" data-permalink=\"https:\/\/suigenerisbrewing.com\/index.php\/2022\/11\/02\/this-is-the-wild-brettanomyces-your-are-looking-for\/ox_anox_plate\/\" data-orig-file=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/ox_anox_plate.png?fit=1205%2C898&amp;ssl=1\" data-orig-size=\"1205,898\" data-comments-opened=\"1\" data-image-meta=\"{&quot;aperture&quot;:&quot;0&quot;,&quot;credit&quot;:&quot;&quot;,&quot;camera&quot;:&quot;&quot;,&quot;caption&quot;:&quot;&quot;,&quot;created_timestamp&quot;:&quot;0&quot;,&quot;copyright&quot;:&quot;&quot;,&quot;focal_length&quot;:&quot;0&quot;,&quot;iso&quot;:&quot;0&quot;,&quot;shutter_speed&quot;:&quot;0&quot;,&quot;title&quot;:&quot;&quot;,&quot;orientation&quot;:&quot;0&quot;}\" data-image-title=\"ox_anox_plate\" data-image-description=\"\" data-image-caption=\"\" data-large-file=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/ox_anox_plate.png?fit=800%2C596&amp;ssl=1\" src=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/ox_anox_plate.png?resize=300%2C224&#038;ssl=1\" alt=\"wild brettanomyces project\" class=\"wp-image-2940\" srcset=\"https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/ox_anox_plate.png?resize=300%2C224&amp;ssl=1 300w, https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/ox_anox_plate.png?resize=1024%2C763&amp;ssl=1 1024w, https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/ox_anox_plate.png?resize=768%2C572&amp;ssl=1 768w, https:\/\/i0.wp.com\/suigenerisbrewing.com\/wp-content\/uploads\/2022\/10\/ox_anox_plate.png?w=1205&amp;ssl=1 1205w\" sizes=\"auto, (max-width: 300px) 100vw, 300px\" \/><figcaption class=\"wp-element-caption\">Characteristic white vs blue <br>colonies on bromocresol green.<\/figcaption><\/figure>\n<\/div>\n\n\n<p>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 &#8220;tentative identification&#8221; 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 &#8211; see image to the left). 3) I screened the colonies that passed criteria 1 &amp; 2 by microscopy, analyzing them for a <em>Brettanomyces<\/em>-like morphology. Slow growing, white yeast colonies, with <em>Brett<\/em>-like morphology, were assumed to be <em>Brettanomyces<\/em> for the sake of validating this approach. The goal here is quick scoring, not in-depth analysis.<\/p>\n\n\n\n<hr class=\"wp-block-separator has-alpha-channel-opacity\"\/>\n\n\n\n<h2 class=\"wp-block-heading\">Results<\/h2>\n\n\n\n<p class=\"has-small-font-size\"><strong>Table 4<\/strong>:<\/p>\n\n\n\n<div class=\"wp-block-columns is-layout-flex wp-container-core-columns-is-layout-9d6595d7 wp-block-columns-is-layout-flex\">\n<div class=\"wp-block-column is-layout-flow wp-block-column-is-layout-flow\" style=\"flex-basis:60%\">\n<figure class=\"wp-block-table is-style-stripes has-small-font-size\"><table class=\"has-fixed-layout\"><thead><tr><th class=\"has-text-align-center\" data-align=\"center\">Test<\/th><th>Eth\/CHex\/<br>AA*<\/th><th>Growth**<\/th><th>% White***<\/th><th>Brett Morphology<br>****<\/th><\/tr><\/thead><tbody><tr><td class=\"has-text-align-center\" data-align=\"center\">1<\/td><td>2\/50\/25<\/td><td>High<\/td><td>&lt;10%<\/td><td>&lt;5%<\/td><\/tr><tr><td class=\"has-text-align-center\" data-align=\"center\">2<\/td><td>2\/100\/50<\/td><td>Low<\/td><td>~50%<\/td><td>~50%<\/td><\/tr><tr><td class=\"has-text-align-center\" data-align=\"center\">3<\/td><td>4\/50\/50<\/td><td>Medium<\/td><td>~50%<\/td><td>~50%<\/td><\/tr><tr><td class=\"has-text-align-center\" data-align=\"center\">4<\/td><td>4\/100\/25<\/td><td>Low<\/td><td>~25%<\/td><td>~50%<\/td><\/tr><\/tbody><\/table><\/figure>\n<\/div>\n<\/div>\n\n\n\n<ul class=\"wp-block-list\">\n<li class=\"has-small-font-size\">* Ethanol (%)\/Cycloheximide (mg\/L)\/acetic acid (mM)<\/li>\n\n\n\n<li class=\"has-small-font-size\">** Relative number of non-mould colonies<\/li>\n\n\n\n<li class=\"has-small-font-size\">*** Portion of colonies that are white (e.g. potentially <em>Brettanomyces<\/em>)<\/li>\n\n\n\n<li class=\"has-small-font-size\">**** Portion of white colonies with <em>Brettanomyces<\/em>-like cells<\/li>\n<\/ul>\n\n\n\n<p>The analysis here is somewhat interesting. Firstly, alcohol  concentration doesn&#8217;t correlate highly with <em>Brettanomyces <\/em>recovery, but does reduce the number of extraneous colonies. The second is that acetic acid enriches for yeast with <em>Brett<\/em>-like morphology more than the other agents, whereas cycloheximide seems to control the total number of yeast colonies (<em>Brett +<\/em> non-<em>Brett<\/em>). What&#8217;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 <em>Brettanomyces<\/em> colonies to form, during which time mould can nearly cover the plate. <\/p>\n\n\n\n<p>Sadly, this is as far as I got in 2024&#8230;as I mentioned in my <a href=\"https:\/\/suigenerisbrewing.com\/?s=Fifty+Meter+Beer+Project\">50 meter beer project<\/a> videos, last year was a crazy year outside of the brewery (silly job, getting in the way of my hobbies). But it wasn&#8217;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, <a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/9802222\/\">but does not affect the growth of the family of yeasts to which <em>Brettanomyces<\/em> belongs<\/a>. So I have a plan for 2025&#8230;and hopefully the end is in sight for this project:<\/p>\n\n\n\n<ol class=\"wp-block-list\">\n<li>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).<\/li>\n\n\n\n<li>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.<\/li>\n<\/ol>\n\n\n\n<p>With luck, this will yield a good population of wild <em>Brettanomyces<\/em> for further investigation! I&#8217;ve already started preparing medium for 2025, so hopefully there will be instagram posts (or even blog posts) soon!<\/p>\n","protected":false},"excerpt":{"rendered":"<p>My longtime readers will know about my Wild Brettanomyces Project &#8211; my multi-year quest to try and identify the environmental<\/p>\n","protected":false},"author":1,"featured_media":2934,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"jetpack_post_was_ever_published":false,"_jetpack_newsletter_access":"","_jetpack_dont_email_post_to_subs":false,"_jetpack_newsletter_tier_id":0,"_jetpack_memberships_contains_paywalled_content":false,"_jetpack_memberships_contains_paid_content":false,"footnotes":"","jetpack_publicize_message":"","jetpack_publicize_feature_enabled":true,"jetpack_social_post_already_shared":true,"jetpack_social_options":{"image_generator_settings":{"template":"highway","default_image_id":0,"font":"","enabled":false},"version":2}},"categories":[16,14],"tags":[160,205,178,177],"class_list":["post-3535","post","type-post","status-publish","format-standard","has-post-thumbnail","hentry","category-adventures","category-yeast-wrangling","tag-brettanomyces","tag-brewing-science","tag-hunting-wild-yeasts","tag-wild-yeast"],"yoast_head":"<!-- This site is optimized with the Yoast SEO plugin v27.3 - https:\/\/yoast.com\/product\/yoast-seo-wordpress\/ -->\n<title>Wild Brettanomyces Project - 2024 Update - Sui Generis Brewing<\/title>\n<meta name=\"description\" content=\"The Wild Brettanomyces Project continues! 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