Completely attenuated 100% Lactobacillus fermentations are not possible

[lshaner]

People may have seen me post in other threads that fully attenuated (defined as >70% apparent attenuation) 100% Lactobacillus fermentations are not possible and those reported as such were in reality a result of unintentionally introducing yeast. To prove that point, I performed an experiment at Omega Yeast Labs to address the question. Here is what I did: I obtained the two Lacto strains from White Labs (delbrueckii and brevis) and Wyeast (buchneri and brevis), and the Omega L. plantarum strain. I grew them on AOAC plates (a Lactobacillus medium) supplemented with cycloheximide. Propagated the lacto in liquid AOAC at ~90F (no stirring) to maximum cell density (achieved in about 3 days). Pitched 200 mL of these cultures into 2 liters of autoclave-sterilized unhopped wort with a starting gravity of 1.037. Incubated for 2 weeks at ~90F, periodically checking gravity and pH.

Final Results:
WL brevis: pH 3.14, 1.035
WL delbrueckii: pH 3.72, 1.033
WY brevis: pH 3.72, 1.035
WY buchneri: pH 3.61, 1.034
OYL plantarum: pH 3.17, 1.036

None of the cultures, whether heterofermentative or homofermentative, produced a significant gravity drop. None produced a krausen or obvious CO2 evolution.

We're going to send the samples off to be tested for alcohol levels so we can get an idea of how much alcohol the heterofermentative strains produce. I'll update this post when we have those results.

Here is the take home message: If you see significant krausen development or gravity drops in your Lacto starters or kettle sours, you have a yeast infection. It's not a result of the Lactobacillus.

 

[ol-hazza]

This is good to know.

Roughly how long did it take to drop these points?

 

[lshaner]

First check was at one week. Very little movement from week 1 to final check at week 2.

 

[Calder]

Something is wrong, when the Homofermentative strain has the lowest FG (combined with the highest ph).

I've done several BWs using probiotics. When using L. Acidopholis (homo-fermentative), I have had little change in gravity over a week at 100F, but had a lot of souring. When using a probiotic with a mixture of Lacto strains, I have had it go from 1.036 to 1.012 twice.

All batches were made, and treated, the same. Something in the mixture of lacto strains went to work on the sugars.

I'm not convinced by your experiment, as noted above, something is wrong since the Homo strain (delbrueckii) had the lowest FG, and my own experience says different (although it is possible there is something in the mix that is not lacto that is doing the work - but it would need to survive the acidity).

If there were trace amounts of yeast present, I think a BW gets too acidic too quickly for it to have any affect on the gravity outcome.

 

[lshaner]

Something is wrong, when the Homofermentative strain has the lowest FG (combined with the highest ph).

I've done several BWs using probiotics. When using L. Acidopholis (homo-fermentative), I have had little change in gravity over a week at 100F, but had a lot of souring. When using a probiotic with a mixture of Lacto strains, I have had it go from 1.036 to 1.012 twice.

All batches were made, and treated, the same. Something in the mixture of lacto strains went to work on the sugars.

I'm not convinced by your experiment, as noted above, something is wrong since the Homo strain (delbrueckii) had the lowest FG, and my own experience says different (although it is possible there is something in the mix that is not lacto that is doing the work - but it would need to survive the acidity).

If there were trace amounts of yeast present, I think a BW gets too acidic too quickly for it to have any affect on the gravity outcome.

Do you have a sample of the Berliner that went from 1.036 to 1.012? I would be happy to look under the scope at it for you. There will be yeast there.

The conventional wisdom that yeast struggles at pH under 3.5 is not entirely accurate. We have done kettle sours at pH 3.1 that yeast have fermented out in 3-4 days. All of our kettle soured beers have been under 3.5. Never had any problems.

As far as the homofermentative strain dropping the pH further -- Lacto doesn't only make lactic acid out of sugars. It generates its biomass from that sugar, secondary metabolites, etc. I'm not sure what else I could do to convince you. I'm a Ph. D. microbiologist with pH meters, autoclaves, knowledge of aseptic culturing techniques, and a microscope at my disposal. This is what I do for a living.

 

[AK7007]

None produced a krausen or obvious CO2 evolution.

Obvious CO2 as in airlock activity, or as in durham tubes don't show it either? I'd be interested how they would manage a gravity drop of any kind without CO2 evolution, metabolism-wise. Does lactate have a large enough discrepancy in it's density contribution compared to dissolved simple sugars that you can see it on a hydrometer? I guess they are ripping a H2O off of whatever sugars to make lactate, but IDK the specifics regarding density.

Are the heterofermentative strains just not putting off that much EtOH to lend large density changes? They are producing CO2 if they are producing ethanol. (EDIT: what I'm getting at is, that if they are obligate hetero strains, they produce a mole of CO2 and EtOH for every mole of lactate produced. The drop in pH says they are probably producing lactate, which means both EtOH and CO2, and some corresponding gravity change. Or are they not obligate?)

 

[lshaner]

Obvious CO2 as in airlock activity, or as in durham tubes don't show it either? I'd be interested how they would manage a gravity drop of any kind without CO2 evolution, metabolism-wise. Does lactate have a large enough discrepancy in it's density contribution compared to dissolved simple sugars that you can see it on a hydrometer? I guess they are ripping a H2O off of whatever sugars to make lactate, but IDK the specifics regarding density.

Are the heterofermentative strains just not putting off that much EtOH to lend large density changes? They are producing CO2 if they are producing ethanol. (EDIT: what I'm getting at is, that if they are obligate hetero strains, they produce a mole of CO2 and EtOH for every mole of lactate produced. The drop in pH says they are probably producing lactate, which means both EtOH and CO2, and some corresponding gravity change. Or are they not obligate?)

As in no visible bubbling in the wort. Didn't put an airlock on -- just foil. Again -- they don't only make lactate from the sugars. They also produce biomass (all the proteins and lipids, etc of the cell).

Heterofermentative strains also have a homofermentative pathway operating at the same time. So lactate, CO2 and EtOH are not equimolar in a heterofermentative strain. You'll get more moles of lactate than CO2 or EtOH. Check out brevis in the first table in this post to see what I mean: http://phdinbeer.com/2015/04/13/physiology-of-flavors-in-beer-lactobacillus-species/

 

[AK7007]

As in no visible bubbling in the wort. Didn't put an airlock on -- just foil. Again -- they don't only make lactate from the sugars. They also produce biomass (all the proteins and lipids, etc of the cell).

Are you saying that gravity drops in the case you see are likely attributable to biomass production and lactate is not relevant? Does that mean you centrifuge out the bacteria? Are you set up to dry the pellet and measure weight? I'm just wondering if a hydrometer normally shows density of the actual liquid with suspended bacteria, or if they interfere at all.

(EDIT: even if you attributed 100% of the 2 point drop in gravity to biomass formation, that comes out to something like 4g of bacteria that grew up in 2L. That sounds low to me, but my experience is growing up stuff for protein work, so who knows, 4g might be a ton - nvmd, just looked and found that yogurt people get ~5g/l when they are growing up starters)

Heterofermentative strains also have a homofermentative pathway operating at the same time. So lactate, CO2 and EtOH are not equimolar in a heterofermentative strain. You'll get more moles of lactate than CO2 or EtOH. Check out brevis in the first table in this post to see what I mean: http://phdinbeer.com/2015/04/13/physiology-of-flavors-in-beer-lactobacillus-species/

That table is reported in g/L. The MW of lactate is ~double (90 vs 46) that of ethanol. So, when they report that brevis is producing 2.5X as much lactate as ethanol, it's skewed pretty far towards pentose phosphate vs glycolysis, since theoretically if it was all pentose phosphate, you would produce ~2X as much lactate than ethanol in terms of raw g/l. This means that in the case of the L. brevis cited, you would expect ~.75-1.4 g/l of CO2 produced as well since CO2 and ethanol have around the same molecular weight. How many moles of lactate are being produced to get down to a pH of 3.14 in your case? You should be expecting a little less CO2 produced than that moles-wise, and a corresponding gravity change. Does it actually track like that though?

Similar to you, I also really doubt that people are getting 100% lacto strains to do these crazy attenuations, but without breaking out the calculator and doing the math, a gravity drop of .002 for your brevis strain just seemed low considering the final pH. No idea if that's a founded concern considering I don't know the original pH of your wort or what the buffering capacity of it was. I personally probably would have titrated your starting wort with lactic acid and measured gas evolution via liquid displacement so I could roughly track what the bacteria were doing using pH, gas, and biomass as a readout, but I also get that your goal was just to show people that lacto doesn't take 1.040 wort down to 1.010.

Also, as an aside - you should suggest to Omega Labs setting up a small distillation apparatus for measuring alcohol content when you want to know real attenuation since they are pretty cheap. I don't know if it's actually cheaper for you to send your stuff out to be tested, (I think Mike Tonsmire said like $75 for the test in a blog post once, though don't quote me) but poking around local universities, I'm pretty sure you could end up setting up your own rig for free if it came to that.

 

[Calder]

Do you have a sample of the Berliner that went from 1.036 to 1.012?

Sorry, all consumed. And it was two batches that acted the same with the same source of Lacto. Maybe there was something else in there that lowered the FG, but to my mind it was lacto.

The conventional wisdom that yeast struggles at pH under 3.5 is not entirely accurate. We have done kettle sours at pH 3.1 that yeast have fermented out in 3-4 days. All of our kettle soured beers have been under 3.5. Never had any problems.

But have you started with just a few (million) cells. Using clean fermenters, there is very little live yeast cells. It's going to take a long time too make a decent colony that is going to have any significant affect on the gravity, and at a low ph will take a long time ...... if ever.

As far as the homofermentative strain dropping the pH further -- Lacto doesn't only make lactic acid out of sugars. It generates its biomass from that sugar, secondary metabolites, etc.

Question: Prior to conducting the experiments, did you predict the Homo Lacto would have the lowest FG? I suspect not, otherwise you would have stated it up-front. Do you really have a solid scientific explanation for it (since you are a Phd I would expect some precision), or are you just trying to wave hands with the biomass theory.

. I'm a Ph. D. microbiologist with pH meters, autoclaves, knowledge of aseptic culturing techniques, and a microscope at my disposal. This is what I do for a living.

I'm just a BSc Engineer with 30 years experience. I have several Phd's working for me, and I know from experience they do not know 'everything'.

I'm not convinced by your 'declaration' of lacto fermentability based on the very limited data you have provided. I think you need to run a lot more tests with more varieties of lacto (I don't think the WLP or Wyeast varieties are very aggressive).


I want to add: I don't know that you are wrong. My experience and many others would indicate that hetero lacto does lower FG considerably. Your single experiment vs. lots of others' experience (including my own) would tend to believe the collective experience.

The explanation of 'produces biomass' ... and that explains it all, rings hollow.

Sorry if I'm being a pain, but I question everything that is different from conventional wisdom. If you are right, we want to know.

 

[AK7007]

I question everything that is different from conventional wisdom. If you are right, we want to know.

I tend to question everything, conventional and non-conventional thought, but also am skeptical of what the OP says because it does not have a logical explanation. I initially didn't like their self-appeal to PhD authority since I'm turing in my graduation papers for a biosci PhD in a few weeks, and one of the things that I have learned from my training is that you have to back everything up with multiple layers of data and explain why you got what result you got, rather than the "trust me, I'm a doctor" line. In this case, that would mean tracking the sugars in the wort. (or at least, that's what I would think to do as a biochemist)

I'm not willing to throw out the idea that a hetero strain produces ethanol and CO2 resulting in a gravity drop. If it's a small amount, that's fine, but the OP's finding seems extreme. Quick, very sour beers are ~1% lactic acid according to Mike Tonsmire's blog. (Who knows, could be wildly wrong, which is why I was so interested in knowing how much lactic acid was in the OP's sample) That would mean that a hetero strain is also putting out ~1% ethanol, and a gravity drop of 7-8 points. That's more than the OP is citing, and less than you are citing.

I tend to think there's something wrong with what both of you are doing regarding the hetero strains. Either he is not seeing hetero fermentation, hetero strains are putting out more than ~1% lactic acid in your case, or you have some random bugs coming along and producing ethanol.

The homo strains are interesting, what with the lowest gravity/highest pH. I would really like to see the biomass idea validated by weighing the cell pellet.

 

[lshaner]

I tend to question everything, conventional and non-conventional thought, but also am skeptical of what the OP says because it does not have a logical explanation. I initially didn't like their self-appeal to PhD authority since I'm turing in my graduation papers for a biosci PhD in a few weeks, and one of the things that I have learned from my training is that you have to back everything up with multiple layers of data and explain why you got what result you got, rather than the "trust me, I'm a doctor" line. In this case, that would mean tracking the sugars in the wort. (or at least, that's what I would think to do as a biochemist)

Look, I don't have a complete explanation of what happened to every sugar molecule in this experiment but that is missing the point of this experiment. And I'm certainly not trying to just toss my credentials around. I was just trying to show that I'm not a dope and I understand how to run a controlled experiment. My only goal here was to prove that you could not take any of the commonly commercially available Lacto strains and completely attenuate wort. Do these results not show that? There are many instances of people on this site and others saying a 100% Lacto pitch of the strains used in this experiment attenuate a wort from 1.040 to 1.010. Why is it that the people making these claims are believed over the results I demonstrated? I would like them to look at a sample under the microscope and prove to me that there isn't yeast in there.

And I avoided saying this in the initial post, but two of the commercial samples had yeast contamination in the commercial packaging as verified by taking a sample straight out of the container and looking at it under the microscope. That is why I plated on AOAC + cycloheximide (allows one step purification of yeast contamination). Seems like that's the logical explanation for so many claims of "100 % Lacto" fermentations, is it not?

 

[ja09]

My only goal here was to prove that you could not take any of the commonly commercially available Lacto strains and completely attenuate

My experience with wlp677 has been the same as Oldsock's: http://www.themadfermentationist.com/2012/06/100-lactobacillus-berliner-weisse.html?m=1

 

[AK7007]

My only goal here was to prove that you could not take any of the commonly commercially available Lacto strains and completely attenuate wort.

My post before the one you quoted acknowledged that, and I agree with the statement that you aren't going to get hetero fermentation to produce the 3-4% abv that is required for full attenuation, mostly because they would have to make too much lactate.

It's just that your experiment's results don't make sense unless 1. Your delbrueckii produced a lot of biomass and not much lactate, which could be, but then it's energy requirements must be kinda low compared to other strains since they are getting their ATP from lactate production. It's just funny, and would be interesting to look into. 2. Your brevis failed to produce much ethanol. Again, possible - but not in line with published stuff. Could be interesting if true.

I agree with your statements regarding lacto fermentations and attenuation, because the biochemistry and math says they are probably right. I don't think that your experiment says anything specific beyond that though, and you were speculating when posters like me were asking questions.

As an aside, I think you'll find contamination to be a common theme among commercial pitches. I've found it, and I'm just a grad student trying to bank all my brewing cultures. Talking to people in the know, part of the issue is that for whatever reason, there is resistance towards using antibiotics to select for what you actually want among old-school beverage (wine and beer) people. As a rule now, when I get new things from White Labs or Wyeast, I streak on selective plates before banking. Related:

Why is it that the people making these claims are believed over the results I demonstrated? I would like them to look at a sample under the microscope and prove to me that there isn't yeast in there.

Two things: 1. People have huge trust built up in white labs and wyeast over the years, so when they are told that something only contains lacto, they implicitly believe it without looking for themselves. (also, most can't look for themselves) 2. You are doing work that is in direct contradiction to the statements being made by white labs and wyeast, but people don't have a long history of trust built up with you. Unfortunate as it is, you therefore need to work extra hard and explore every avenue in order to convince people that your results are right - it's not on them to convince you. You say you demonstrated something in the OP, I say that you didn't exactly provide that much info. pH and gravity aren't a complete picture.

I agree with you about these types of fermentations, and yeast contamination from white labs or wyeast is unsurprising to me - but what you posted in the OP wasn't exactly comprehensive, because it says "hey, 75% attenuation doesn't happen" without also saying exactly what is happening instead. That's going to be a problem when people have been taking white labs and wyeast at their word that they are able to get big gravity drops with hetero fermentations.

 

[TheGarv]

Much of the above strikes me as pretty obtuse, and some of it as a nice example of confirmation bias.

Theoretically speaking, it's always struck me (and a lot of other people who know a whole lot more about microbiology than I do) that 100% lactobacillus fermentations should not be possible, certainly not in the way claimed by people who say they've done them, given the limited tolerance of low pH by lactobacillus, and the low amount of sugar required to very significantly drop the pH.

The burden of proof for this stuff is certainly on people who think 100% lactobacillus fermentation is possible to carry out a proper experiment. You need to do some fermenting under controlled conditions, and to examine the resulting liquid for yeast. If someone does that and finds no yeast, I'd be very surprised, but it would be worth looking into. Armchair speculation about the conditions you reckon yeast will sufficiently multiply under is not a substitute for this.

This is especially so when it's well known that commercial bacterial cultures from, for example, White Labs, are fairly frequently contaminated with yeast (you can google this, if you doubt me - a good number of people have put samples under the microscope).

Without actually having looked at your finished beer scientifically, on what basis do people think their beer is 100% lactobacillus? Is it just assuming that the sample was clean and that your homebrew set-up and process is sanitary? Because that seems a seriously weak reason.

Lance's experiment strikes me as pretty convincing, and if anyone is unsure, they should try to replicate it and see what happens, or, at the least, look at a '100% lacto' beer under a microscope.

The low pH/high FG thing for the homofermentative strain strikes me as a red herring. What do you think could have gone wrong here that affects the result, or tells in favour of the possibility of 100% lacto fermentation?

 

[Warthaug]

Like Garth said, this is an interesting thread and a great example of confirmation bias. The concept of a 100% lacto fermentation reaching full (>70%) attenuation has always been somewhat of a joke. To be blunt, it is a biological impossibility.

Let me repeat that, it is a biological impossibility.

Acid tolerance in bacteria is determined by two phenomena; chemical resistance (i.e. having a surface which is non-reactive with acids) and something called the proton motive force. For acid tolerant organisms, the latter is the key thing to understand when talking about their ability to survive low pH. The proton motive force is used by all bacteria for a number of purposes; in the case of Lactobacilli, it is used to "force" important metabolites such as amino acids and nucleic acids from the extracellular environment to the cytosol where they can be used for protein and DNA synthesis. For some oxygen-utilizing species (including us), it can also be used to generate energy.

The way the PMF works is fairly simple, protons (acid "atoms") are pumped by energy-consuming pumps out of the cell and into the extracellular space. This acidifies the environment and alkalises the cytosol, and in the process creates an electrochemical gradient which can then be harnessed for energy-dependent transport of materials in/out of the cell. While a generally effective system, it has one fatal flaw; the more acidic the extracellular environment, the more energy it takes to pump protons out. This puts a finite limit on the extracellular pH upto which this gradient can be maintained. The theoretical maximum energy, determined by the hydrolysis energy of ATP (the main cellular energy source) and assuming a 100% efficient pump is ~-31kJ/mol, which works out to a pH of ~1.2. But no living organism has a 100% efficient pump, plus most have membranes which are a little leaky when it comes to protons.

A few extremophile bacteria have pumps that operate with 90-98% efficiency, and have modified their cellular membranes to minimize proton leak, allowing them to survive in pH's of 2.0 - 1.3. But they are rarities; the vast majority of bacteria have pumps that operate at 10-30% efficiency and leaky membranes, limiting acid tolerance to 5.0-3.0. The efficiency of Lactobacilli proton pumps is well established, and they can maintain an PMF down to pH's of 2.8 to 3.4, depending on species and strain. That is equivalent to ~0.8% lactic acid, or the quantity of lactic acid produced by Lactobacill after processing a quantity of sugar equivalent to a gravity drop of ~2-6 points (range depends on homo/heterofermentation, and on fraction of carbon used for biomass generation).

Compare that to what would be required for complete attenuation; a drop of ~30 gravity points. At that point you're at ~6% lactic acid by volume (for a homofermentative strain, 3-4% for heterofermentative), which would produce a pH of 1.9 to 4.2; both well into the range which would be acutely lethal for Lactobacilli.

In other words, if you're getting full attenuation with a lacto-only ferment you've got contamination.

Bryan

 

[wardens355]

Like Garth said, this is an interesting thread and a great example of confirmation bias. The concept of a 100% lacto fermentation reaching full (>70%) attenuation has always been somewhat of a joke. To be blunt, it is a biological impossibility....

Thanks for the interesting read. I assume one could maintain a pH (with chemical addition) within the tolerance of the organism throughout fermentation and force higher attenuation that way, however impractical that may be.

 

[sweetcell]

I want to add: I don't know that you are wrong. My experience and many others would indicate that hetero lacto does lower FG considerably. Your single experiment vs. lots of others' experience (including my own) would tend to believe the collective experience.

right, but the problem with the collective experience is that it wasn't done under controlled, scientific conditions. contamination is quite likely. sometime external contamination isn't even required, some commercial lactos come with yeast:

And I avoided saying this in the initial post, but two of the commercial samples had yeast contamination in the commercial packaging as verified by taking a sample straight out of the container and looking at it under the microscope. That is why I plated on AOAC + cycloheximide (allows one step purification of yeast contamination). Seems like that's the logical explanation for so many claims of "100 % Lacto" fermentations, is it not?

 

[berebrando]

Fantastic thread and thank you for posting your results.

Anecdotally, in the last six months I have seen differences in the purity of Homebrew-sized pitches compared to commercial-sized pitches of bacteria. Homebrew bacteria pitches in my experience have been infected with yeast (that is, they ferment and produce very little acidity) while commercial bacteria pitches do not ferment and produce significant acidity. I haven't looked at anything under a microscope, but when two identical worts are inoculated with the same bacteria (per the label) produce dramatically different results, I question the purity of the culture.

Anyways, thanks for posting, and thanks to all who have contributed here! Amazing dialogue.

 

[PLOVE]

Great stuff guys! Some of the most intellectually stimulating talk on brewing science I've read in some time. Way to keep things focused on the science and not personal attacks.

It seems that the experiment we need to do would be to: (a) take a look at these reported pure cultures under a microscope and rule out a yeast contamination, and (b) plate out on appropriate medium to find out what Wyeast and WL are actually including as pure culture (or not).

Very nice work by the OP to isolate pure cultures and run the experiment. In my view, the lack of a drop in gravity is the primary finding. Explanations aside, it's a strong result showing pure lacto cultures can't get the job done without some help. I'm betting, like most of us, that pitching the pure smack pack would have a very different result.

Enjoying this thread immensely. . .
Pete

 

[AK7007]

Compare that to what would be required for complete attenuation; a drop of ~30 gravity points. At that point you're at ~6% lactic acid by volume (for a homofermentative strain, 3-4% for heterofermentative), which would produce a pH of 1.9 to 4.2; both well into the range which would be acutely lethal for Lactobacilli.

Bryan

One quibble: homofermentative strains shouldn't drop gravity at all besides biomass production. Even with impossible complete attenuation, the apparent gravity can't go down 30 points.

That is equivalent to ~0.8% lactic acid, or the quantity of lactic acid produced by Lactobacill after processing a quantity of sugar equivalent to a gravity drop of ~2-6 points (range depends on homo/heterofermentation, and on fraction of carbon used for biomass generation).

I need to go check my math; I had that amount of lactic acid at a slightly higher gravity drop, (~6-8 points) but was literally clicking the numbers on the computer calculator and not writing out my work.

As a side note: beer convention is super annoying when we are actually talking about metabolism. Conversion between measures of volume and weight are bad enough, but the actual metabolism and pH makes the most sense in molarity. The final gravity drop I was calculating in terms of CO2 evolution in molarity and back converting to grams lost from solution. Probably too rough a calculation.

 

[Warthaug]

One quibble: homofermentative strains shouldn't drop gravity at all besides biomass production. Even with impossible complete attenuation, the apparent gravity can't go down 30 points.

True, but I was intending that to be interpreted as the consumption of that many points worth of sugars; regardless of the actual gravity change.

I need to go check my math; I had that amount of lactic acid at a slightly higher gravity drop, (~6-8 points) but was literally clicking the numbers on the computer calculator and not writing out my work.

The values I gave are for unbuffered solutions, so real-world pH drops may be less dramatic given an equivalent consumption of sugars.

B

 

[Warthaug]

(b) plate out on appropriate medium to find out what Wyeast and WL are actually including as pure culture (or not).

There are numerous reports of this occurring; indeed, for my yeast bank I've found myself purifying commercial lacto's right out of the tube/smackpack; i.e. the unopened "bacteria" tubes from the manufacturer had yeast contaminants.

Lance took care of that in his experiment by plating the lacto on a plate not permissive to yeast growth, picking a single colony (i.e. a genetically pure strain of the lacto), and then running that colony through a starter that was not permissive to yeast growth. That's about as pure a strain as you can get...and we all know what his results were.

B

 

[dantheman13]

We've been tracking/discussing this on MTF as well; documented Lance's findings and updates here: http://www.milkthefunk.com/wiki/100%_Lactobacillus_Fermentation#Results

The WLP677 produced alcohol, signifying that it underwent heterolactic fermentation. I wouldn't be surprised if it is (another) misidentified species.

 

[AK7007]

The WLP677 produced alcohol, signifying that it underwent heterolactic fermentation. I wouldn't be surprised if it is (another) misidentified species.

Okay, this is interesting. Multiple older papers cite delbrueckii as strictly homofermentative, unable to ferment pentose sugars. The published genome for delbrueckii bulgaricus isolate ND02 though has an alcohol dehyrogenase, phosphoketolase, and phosphogluconate dehydrogenase. I kind of assumed that those are the key steps to produce ethanol, right? Make a pentose, split it, and then convert acetylaldahyde to ethanol. Looking at the genome annotation paper though, they note that the pentose phosphate pathway is incomplete in the strain, but it still has pentose transporters to get them into the cell. I can't figure out which pentose phosphate enzyme is missing looking at UniProt.

I'm at the limit of caring though, since it's all moot without a definitive species ID. Maybe White Labs has some random delbrueckii that has acquired the last bits to perform heterofermentative metabolism, maybe it's the wrong species, maybe it's a mixture of species.

Is Omega Labs going to do 16s sequencing for these suckers?

Part of me hopes this all becomes moot and White Labs publishes illumina sequences for everything. Part of me isn't holding my breath, since who knows when that is/if the wild yeast and bacteria are being included, and we won't have expression patterns anyways.

EDIT: Oh, and the alcohol production by each strain really helps with the whole pH/gravity/whatever stuff. Thanks for publishing that!

 

[lshaner]

Okay, this is interesting. Multiple older papers cite delbrueckii as strictly homofermentative, unable to ferment pentose sugars. The published genome for delbrueckii bulgaricus isolate ND02 though has an alcohol dehyrogenase, phosphoketolase, and phosphogluconate dehydrogenase. I kind of assumed that those are the key steps to produce ethanol, right? Make a pentose, split it, and then convert acetylaldahyde to ethanol. Looking at the genome annotation paper though, they note that the pentose phosphate pathway is incomplete in the strain, but it still has pentose transporters to get them into the cell. I can't figure out which pentose phosphate enzyme is missing looking at UniProt.

I'm at the limit of caring though, since it's all moot without a definitive species ID. Maybe White Labs has some random delbrueckii that has acquired the last bits to perform heterofermentative metabolism, maybe it's the wrong species, maybe it's a mixture of species.

Is Omega Labs going to do 16s sequencing for these suckers?

Part of me hopes this all becomes moot and White Labs publishes illumina sequences for everything. Part of me isn't holding my breath, since who knows when that is/if the wild yeast and bacteria are being included, and we won't have expression patterns anyways.

EDIT: Oh, and the alcohol production by each strain really helps with the whole pH/gravity/whatever stuff. Thanks for publishing that!

We're not sequencing 677. 677 is either an unusual delbrueckii, like you note, or it's misidentified. That strain blows any way you slice it so I'm not going to spend money in having it sequenced to assuage my curiosity.

 

[Warthaug]

Okay, this is interesting. Multiple older papers cite delbrueckii as strictly homofermentative, unable to ferment pentose sugars. The published genome for delbrueckii bulgaricus isolate ND02 though has an alcohol dehyrogenase, phosphoketolase, and phosphogluconate dehydrogenase. I kind of assumed that those are the key steps to produce ethanol, right?

Not necessarily.

Alcohol dehydrogenases can run "both ways"; i.e. they can make, or allow an organism to consume, alcohol. Most lactobacilli have multiple ADH genes; the majority of those are for the consumption/breakdown of alcohols. The heterofermentative strains have ADH's which make alcohol under normal biological conditions.

Phosphoketolase is also a multi-function enzyme, and is crucial for the synthesis of the amino acid methionine. So again, this enzyme is present in most lactibacilli (perhaps all; I'm not sure if there are any lacto that are auxotrophic for methionine).

The phosphogluconate is odd; AFAIK its only used in the pentose phosphate pathway. Then again, bacteria exchange genes the way highschool students exchange STD's (by which I mean, a lot), so its possible that particular strain of delbrueckii picked it up somewhere else, but otherwise lacks the enzymes it needs to fully use it. The pentose phosphate pathway requires ~8 enzymes to function; the presence of 3 would not be sufficient.

B