Wednesday, 10 September 2014

One mystery solved...

As I mentioned yesterday I recently developed a persistent infection of part of my brewing setup. Thee batches that passed through my primary fermenter ended up infected (based on aroma/taste/texture); in the final beer I was able to confirm the infection directly by examining a wort sample in which I observed chains of highly elongated rod-shaped bacteria.

Aside from having no idea where the infection came from, I also had no idea what it was. The cell size and length:width ratio were too big for lactobacillus; the shape and formation of chains was inconsistent with both pediococcus and acetobacter. In other words, it isn't likely one of our usual suspects. So I ran a variation of the genomic sequencing method I've used previously to ID yeast, but this time with primers that recognize bacteria - producing the following sequence:

AGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGG
GAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGA
GTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTACCGTTCGAA
TAGGGCGGTACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCC
GCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGC
GGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGG
GGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGA
TGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGA
AAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGC
TAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTG
GGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGG

AGCATGTGGTTTAATTCGAAGCA

For those who don't read DNA, that the sequence of Bacillus subtilis's ribosome. This explains...well pretty much everything. Bacillus are spore producing species and their spores are very nearly indestructible - they can survive transient exposure to bleach, are pretty much impermeable to sanitizers like starsan, and can even survive short period of boiling! Moreover, their spores stick to surfaces like crazy-glue, making them near-impossible to remove. The persistence of Bacillus spores, but killing of the vegetative (growing) form after sanitation, would lead to the situation I've experienced - stuff fermented briefly in the container appears normal until weeks after packaging, while beers fermented for longer periods show signs while still in the primary.

Of course, the question is where the heck did this come from? B. subtilis grows in the soil, and is a normal components of our guts - so it is comforting to think that it may be nothing more than an environmental contaminant. But I don't think that is the source - at the time I was developing a teaching lab - using B. subtilis - for an undergrad course that I am currently running.  I have a sneaking suspicion I may literally have brought my work home with me...

2 comments:

  1. For shame- I can't count the number of safety weenies who told us no food or drink in the lab fridge! Ya'll need one of those signs like we have. The good news is you now have a near-definite source and can fix things accordingly. And nuke from orbit your old bucket and anything else that touched the contaminated brews. Sounds like a right nasty piece of work that bacteria.

    At least now you can have a nice warm and fuzzy sense of closure as you mourn for your lost batches I suppose.
    - Dennis, Life Fermented Blog

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    Replies
    1. At least now you can have a nice warm and fuzzy sense of closure as you mourn for your lost batches I suppose.
      More a sense of shame than closure. You'd think after nearly 20 years in the lab I'd be able to manage the rather plebeian task of not cross-contaminating my cultures.

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