Brett + bacteria = worse, or better

Microbiology has gotten a lot wrong studying yeast and bacteria. We’ve assumed, until quite recently, that if a microbe doesn’t grow in a dish it’s not there. And that a microbe is either on/live/growing or off/dead. And that we can study microbes in isolation — “pure culture” — away from other species in little sterile dishes and expect them to behave normally. In all fairness, microbiologists have sometimes seen these as a problems, but have mostly just gone on this way, writing books about what we think we know.

DNA detection and sequencing technology is showing just how many bugs don’t grow in dishes — “high throughput” technology can document (theoretically) all of the species in a drop of [insert favorite liquid here]. That’s pretty routine these days. And we’re slowly beginning to study how mixtures of microbes — you know, the way they live in the wild — behave in the lab. Wine was a bit ahead of the curve here: microbial enologists have been studying the goings-on of spontaneous and mixed fermentations since the late 1980’s.*

Usually, mixed-microbe studies are about what grows where together. Occasionally, you can predict something more specific with a bit of logic and some scratch paper. That, plus a little knowledge of yeast and bacteria metabolism, leads to an interesting hypothesis: some malolactic fermentation bacteria should make Brett smell worse.

Brettanomyces bruxellensis (aka “Brett,” aka barnyard-stench spoilage yeast) creates its signature aroma by converting hydroxycinnamic acids (HCAs) naturally present in wine to smelly volatile phenols. This is a two-step process. First, an enzyme (a decarboxylase) converts HCA to a vinylphenol. Second, a different enzyme (a reductase) converts the vinylphenol to the volatile ethylphenol, including the Brett signature 4-EP and 4-EG.

But before that can happen, Brett has to be able to get to the HCAs. Many of the HCAs in wine are chemically bound to tartaric acid. Brett can’t use them if they’re bound. The HCA-tartaric acid bond spontaneously and slowly breaks, giving off free HCAs for Brett to use, but there’s theoretically a much bigger pool of pre-stink molecules that need only lose their acid first.

Some lactic acid bacteria — like the ones that commonly perform the malolactic fermentation (MLF) so important to most reds and a lot of white wines — can enzymatically split HCAs from tartaric acid. In theory, that should mean that some (but not all) MLF bacteria are Brett enablers. Wine + bacteria + Brett = worse smell than wine + Brett alone.

Building on previous research, a team at Oregon State University has made that more than a theory. Their recent paper (currently pre-press in AJEV) shows that some commercially available MLF strains make more HCAs available than others, AND that leads to Brett making more 4-EP and 4-EG,

The team only experimented with one strain of Brettanomyces, and they obviously couldn’t test anywhere near all of the MLF strains on the market, but this (plus the multiple studies that have come before it supporting the effects of lactic acid bacteria on HCAs) is strong evidence indeed that winemakers buying commercial bacteria for MLF may have better and worse choices if they’re worried about Brett.

 

*A good open-access (no paywall) example of this kind of research is Granchi and company’s 1999 study here.

Studying sulfur dioxide effects with better DNA technology suggests we may not need much of it

Fast: In a new study using better-than-ever microbiology, 25 mg/L SO2 added after pressing was enough to “stabilize” yeast and bacterial growth during fermentation, and higher concentrations actually seemed to slow fermentation. Inoculating the must with commercial S. cerevisiae had a very similar effect, even without adding SO2, which looks really, really good for no-added-sulfur wines. BUT (and this is a big but) the study only included one wine (a California chardonnay) made in one way, in smallish (19 L) lab volumes. Goodness only knows if their results will generalize, but let’s hope this encourages someone to look.

More: Sulfur dioxide is the single most commonly used winemaking chemical worldwide. That familiarity probably has something to do with our not understanding it better: we know it’s safe, we know how to use it, and so we don’t have much reason to study it.

In all fairness we do understand SO2 well, but microbiology keeps changing. The publishing dynamo* of Nicholas Bokulich and David Mills – responsible for really excellent recent research on how microbes are spread around a working winery over space and time – plus UC Davis wine microbiologist Linda Bisson (and another Davis student and a Japanese collaborator have published a new American Journal of Enology and Viticulture article on how SO2 affects bacteria and yeast populations in fermenting wine.

The question isn’t new, but the technology they’re using is. Short story: better DNA detection techniques let them pick up on the presence of a bigger range of both bacteria and yeast than previous strategies.

Longer story: Microbes in wine (and elsewhere, for that matter) can be “viable but nonculturable” (VBNC), a new idea ten years ago when microbiologists could still think that agar and Petri dishes were a reasonable way of identifying bugs in a sample. Until better DNA technology made clear a serious issue: yeast and bacteria might be stressed out enough by environments (like wine) to not grow on command but still be alive and able to multiply and cause problems, aka VBNC. (The unculturables who won’t grow in dishes at all are trouble, too.) The details of the high-throughput DNA sequencing they used to ensure that VBNC bugs weren’t left out of their survey aren’t important except to note that it lets them detect more microbes than previous studies.

The other great = new element of this study is its looking at multiple SO2 concentrations, from 150 mg/L down to nil. More work for them; more data for everyone. They also included ferments inoculated with commercial S. cerevisiae and not, which ended up being important.

Their results say that the most important factor in determining what grows in fermenting wine seems to be the degree to which a single strain has the opportunity to take over. One way of encouraging dominance is inoculating with commercial yeast: it more or less takes over and overall microbe diversity declines. But another way is adding SO2, which knocks down some microbes and gives tolerant ones (S. cerevisiae strains included) an opening. Adding SO2 and inoculating S. cerevisiae even without SO2 had similar effects on overall microbial diversity. And, moreover, 25 mg/L was enough SO2 to “stabilize” the ferment. In other words, sulfur-free wines may be less risky than winemakers are generally inclined to believe if they inoculate (which plenty of people inclined not to use sulfur are also inclined to avoid).

The obvious problem: one wine, one vintage, one set of processing techniques, and 19 L volumes. All of these are major reasons to question whether these results will hold for any other set of circumstances. pH and a slew of sulfur-binding compounds affect SO2 efficacy. Fermentation temperature, oxygen, clarification, means of harvesting…the list of processing steps important to microbial diversity is too long to list. And it’s well-known that fermentation volume is important to microbial kinetics.

In short? This article is almost certainly more important to wine microbiologists as a methods paper than to winemakers. (It’s not incidental that the methods section abbreviates the winemaking protocol — “grapes were harvested, crushed, and pressed according to standard winemaking procedures,” whatever that means – and uses nearly a full page of text to describe the DNA sequencing technique.) Nevertheless, it may well serve as impetus for more experimentation with low- and no-sulfur wines, and a good reminder that we always have more to learn about SO2.

Even more: find the full paper, with many more details on which specific yeast and bacteria species were detected and when they peaked (unfortunately behind the AJEV‘s lovable paywall) here. Read the full paper if you can; it contains plenty of potentially idea-generating details that I’ve not even attempted to summarize here.

*Hackneyed maybe, but, seriously, what else do you call them? Bokulich’s CV, as a PhD student, could put to shame plenty of tenured professors. When I’m not just feeling horribly inadequate, I’m wondering where this guy will end up post-graduation. Barring his speaking French and fancying living overseas – or starting a lucrative consulting firm – he’s probably lined up to make tenure at Davis in record time. Heck, he probably already qualifies for tenure.

Why stuck fermentations are like Mad Cow Disease

Stuck fermentations — when sugar levels stop dropping and the winemaking process stands still — are one of the more persistently frustrating mysteries in winemaking. Like most winemaking mysteries, we understand part but not all of the situation. Bacterial contamination is one of numerous known causes of sticking: lactic acid bacteria can compete with wine for access to sugar, but it’s also long seemed that something else is going on. Researchers now have a better idea of what that something else is, and it involves prions.** Yes, prions, best known by nearly everyone as the infective agent in bovine spongiform encephalopathy, more fondly known as Mad Cow Disease.

Briefly, bacteria are producing some kind of small signaling molecule that provokes Saccharomyces cerivisiae to shift from preferentially fermenting glucose into alcohol to consuming other energy sources indiscriminately. Bacteria release the molecule, yeast take the molecule up and begin expressing a prion, and in some as-yet-unknown way, the prion jams the mechanism that normally tells yeast to consume only glucose when it has both glucose and other energy sources available. Bacteria don’t tolerate alcohol as well as S. cerivisiae, so it’s in the bacteria’s interest to get the yeast to make less of it. S. cerivisiae can use all manner of different molecules for energy, but a specific control mechanism ensures that it (usually) eats glucose first when glucose is around.

These findings tie into an overwhelming lot of very interesting, very intricate biology, the fullness of which is a bit much to discuss here. But (understanding that there are others), a few reasons why this research matters to scientists and to winemakers stands out.

To scientists:

  • Bacteria and yeast are talking to each other. Or, rather, bacteria are controlling yeast for the bacteria’s benefit. Bacteria produce lots of small messenger molecules — a bit like hormones in the human body — to communicate amongst themselves. But the idea that they use a similar molecule to control the behavior of a different species is exciting. Bacteria probably do this all the time, too, but microbiologists are behind on learning about it because we traditionally study one type of microbe at a time, by itself, in a test tube or beaker. Imagine studying 12 year-old boy behavior by putting lots of 12 year-old boys in a room by themselves and watching them for a week. That’s what we’ve been doing with bacteria. Microbiology as a field is increasingly realizing that there are better ways (which are, of course, more complicated, and therefore harder…)
  • The mechanism involves prions, which are cool because they’re a relatively recent discovery and we’re finding them in places we didn’t see coming. It’s still not clear how they’re working in this setting, but finding out will almost certainly involve learning some new and interesting biology

To winemakers:

  • Winemakers who are adamant about avoiding stuck fermentations are probably also vigilant about trying to keep bacterial contamination out of their wines, so I imagine this news doesn’t change much. Nonetheless, some folk might end up using more sulfur dioxide in an effort to knock down bacteria in ferments that tend toward stickiness.
  • More interestingly, researchers may be able to develop yeast that don’t respond to the bacteria-induced switch, maybe with a mutated form of the prion protein. Non-stick yeast?

**The research is published in two complementary papers (here and here) in the journal Cell and, as happens with particularly interesting stuff like this, the editors have put together a short summary. It’s still pretty dense stuff unless you have a background in molecular microbiology, but you can find it here if you’re interested in the details (and if you have institutional access to the journal).