100% Lactobacillus Fermentation

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100% Lactobacillus Fermentation is the process of using a single pure strain of Lactobacillus to ferment with. This page will primary discuss the results of tests done by Lance Shaner from Omega Yeast Labs, as well as scientific explanations of why achieving full attenuation with Lactobacillus does not appear to be biologically possible. See the Lactobacillus page for more details on Lactobacillus in general.

History

Previously there have been claims of 100% Lactobacillus fermented berliner weisse beers. An example includes Michael Tonsmeire's 100% Lactobacillus fermented berliner wiesse using WLP677. Interestingly, the fermentation looked like a yeast fermentation and the beer was described by Tonsmeire as being lacking in acidity [1]. Commercial examples of beers claiming to be 100% fermented by Lactobacillus once included The Bruery's Hottenroth, however it has been confirmed that Hottenroth also has Brettanomyces in it [2][3]. There have been reports of finding yeast cells in WLP677 vials on MTF [4]. More recently an employee of White Labs explained on MTF that once the WLP677 product is packaged using the PurePitch® technology, yeast contamination in this product should no longer be an issue for the company [5].

See also Lactobacillus fermentation.

Lance Shaner's Experiment

This experiment showed that fully attenuated (defined as >70% apparent attenuation) 100% Lactobacillus fermentations were not possible and those reported as such were in reality a result of unintentionally introducing yeast.

Procedures

Two Lacto strains from White Labs (delbrueckii and brevis) and Wyeast (buchneri and brevis), and the Omega L. plantarum strain were tested. All were grown on AOAC plates (a Lactobacillus medium) supplemented with cycloheximide. All Lacto cultures were cultivated in liquid AOAC at ~90°F (no stirring) to maximum cell density (achieved in about 3 days). 200 ml of each Lactobacillus was pitched into 2 liters of autoclave-sterilized unhopped wort with a starting gravity of 1.037. Each fermentation was incubated for 2 weeks at ~90°F, periodically checking gravity and pH.

Results

Lacto Species/Strain Final pH Appt. Specific Gravity (Anton Paar Alcolyzer) ABV (Anton Paar Alcolyzer)
White Labs L. brevis 3.14 1.03386 0.10%
White Labs L. delbruekii 3.72 1.03255 0.29%
Wyeast L. brevis 3.72 1.03386 0.18%
Wyeast L. buchneri 3.61 1.03279 0.24%
Omega Yeast Labs L. plantarum 3.17 1.03394 0.10%

None of the cultures, whether heterofermentative or homofermentative (all were heterofermentative), produced a significant gravity drop. None produced a krausen or obvious CO2 evolution. All samples were sent off to be tested for alcohol levels using the Anton Paar Alcolyzer test. All strains showed some alcohol production, indicating heterolactic fermentation. The wort was fermented with foil covering the flasks, so it was not an anaerobic environment [6]. Note that the pH readings of this test correspond with the results of Matt Humbard's pH tests with Lacto.

Biological Explanations

Bryan of Sui Generis Blog Biology Explanation

"This is what I would have expected - those claims of complete attenuation always rubbed me the wrong way, and from a scientific point of view, appeared impossible. pH will (eventually) inhibit even the most acid-tolerant of bacteria. Even if the bug is capable of protecting itself from the degradative nature of acids (which many can), at some point the concentration of protons (acid "atoms") will block the bacteria's proton motive force, which is used to "pump" molecules into bacteria (and in the case of some bacteria, to generate energy). Once the proton motive force is gone, the bacteria is effectively dead.

Bacteria setup a proton motive force by pumping protons out of their cytosol, into the extracellular space. This acidifies the extracellular space and alkinalyzes the cytosol. The energy of this gradient can then be used to "force" other molecules into the cell - e.g. Lactobacillus uses this to collect amino acids and DNA for growth and replication. However, the more acidic the environment, the more energy is required to pump additional protons out of the cell. The ability of cells to do this is finite; the theoretical maximum "pump force" that can be exerted is equal to the energy of hydrolysis of one ATP (energy molecule); roughly -31 kJ/mol. The theoretical max acidity that will work against is 1.2, but in reality these pumps are generally less than 30% efficient, so for most species pumps fail at pH's above 3.0. Most Lactobacillus crap out somewhere around 3.2 to 3.0. The pH of a fully attenuated 1.040 wort would be between 1.9 and 2.4 (ignoring buffering effects), depending on whether the Lactobacillus was homo or heterofermentive. So the claim that Lactobacillus can completely ferment wort doesn't seem to be biologically plausible." - Comments by Bryan of Sui Generis Blog [7]

Elke Arendt

Elke Arendt, a scientist that specializes in Lactobacillus, presented her work at the Belgian Brewing Conference 2015. In it she explained that Lactobacillus will only ferment 0.5°P of wort regardless of the gravity of that wort, confirming Shaner's results above. When asked at the end of the presentation why Lactobacillus only ferments ~0.5°P (note that Shaner's experiment shows Lactobacillus fermenting ~1°P, although this may be due to a margin of error since Shaner only performed this experiment once), considering that Lactobacillus ferments maltose and there is plenty of maltose in wort, Arendt responded that she believes that the bacteria inhibits itself and that it reaches max cell density in the wort with relatively little sugar requirements (~16 mins in and ~25 mins in):

See Also

Additional Articles on MTF Wiki

External Resources

References

  1. 100% Lactobacillus Berliner Weisse Tasting. The Mad Fermentationist. 05/27/2013. Retrieved 02/10/2016.
  2. American Sour Beer: Innovative Techniques for Mixed Fermentations. Michael Tonsmeire. 2014.
  3. Conversation on MTF regarding Hottenroth. 10/08/2015.
  4. Conversation about WLP677 on MTF. 01/12/2016.
  5. Conversation about WLP677 on MTF. 01/12/2016.
  6. Conversation with Lance Shaner on MTF regarding his 100% Lacto Fermentation results. 7/21/2015.
  7. Conversation with Bryan of Sui Generis Blog on 100% lacto fermentation. 7/22/2015.