Chocolate has been on my mind as we draw close to Easter. First, because it's a delicious food of the gods (and will be super cheap on Monday!). Second, if you've been paying any attention at all to my articles you're not going to be surprised that microbes are both critical to chocolate as we know it and the reasons they're critical are illustrative of some broader microbial and ecological concepts.

But there is a little difference between this article and my previous ones. Those have generally focused on specific bugs in certain environments. Understanding chocolate is all about understanding an succession of bugs who both change and, in turn, are affected by their environment. In fact, during many parts of chocolate production, the exact bug present doesn't really matter, so long as it performs certain ecological roles.

Perhaps my favorite part of Spring. Source by ivabalkunder CC0.^†

Chocolate botany 101

When I was growing up, I didn't think much about where chocolate came from. I did have cacao nibs, so I suppose that if anyone asked me I'd have said that chocolate must be a lot like coffee, probably some single bean that's roasted and crushed. I wouldn't have been terribly wrong, but I'd have been missing some important details.

First, know that cacao doesn't grow as a single berry or bean, instead it grows as large pods on the Theobroma cacao tree. Inside each of these pods is a gooey white pulp and a bunch of seeds. At this stage, the pulp itself is edible is the only sweet thing, the beans themselves are pretty bitter.

That flavor profile makes sense - for a plant the whole point of fruit is to provide a lure to get some animal to ingest, but not destroy, your seeds so that your genes can be widely dispersed, keeping you from competing with your offspring. While the fruit should be tasty, the seeds should be rugged, unpalatable, or even poisonous (if cracked open). In the case of cacao, the sweet pulp is what is supposed to entice us to eat, while the bitter seeds are supposed to dissuade us from chewing too hard. This strategy works pretty well, until clever primates come along and decide that they really like the flavor - hint, chili peppers aren't delicious on purpose. Of course, Michael Pollan might point out that if this backfires with a species that's prone to cultivate delicious things, the plant might do extraordinarily well.

Cacao tree and opened pod. Source by AoC in public domain.

Something is rotten in the state of Theobroma

To go from bitter seeds to the treat we all know and love, we need a series of microbial helpers. There's actually two different ways fermentation plays into chocolate production - one is pretty much necessary and the other imparts a certain specialized 'zing' to some varieties. Let's tackle them in turn.

A classic case of rotting beans

Remember that gooey white pulp? It turns out to be filled with sugars (~ 15% glucose, fructose, and sucrose), pectin, and some proteins. Bugs love to eat this stuff and they start eating it pretty much as soon as the sterile interior of the pod is exposed to air. At some point in history, someone noticed that their rotting cacao pods tasted pretty great actually^††, and that was the start of a long road towards chocolate as we know it.

Succession in cacao fermentation

Do you remember high school biology class where you learned about ecological succession? Perhaps something about stones getting covered with lichen, and then small plants and mosses invading, followed by a field and ending up with a particular kind of forest?

Ecological succession. Source by LucasMartinFrey under CC3.

The same kind of thing happens during chocolate fermentation - the farmers will pack a bunch of pod pulp and seeds into a ventilated box and over the course of a few days, different types of bugs will grow, eat different things, and change their environment. The changes in the environment, in turn, alters what bugs will thrive.

Fermentation box full of cacao pod pulp and beans. Source by Irene Scott under CC2.

The initial packed pod pulp is acidic (mainly from citric acid), high in a variety of sugars, and not very well aerated. Yeasts, which love all these things are the pioneer organisms in for that system. During the first few days, yeasts are the dominant population, happily munching away at sugars and citric acid, producing ethanol and little CO₂ burps.

Unfortunately for the yeasts, they become a victim of their own success. Eating up the citric acid increases the pH of the pulp and all those burps create lots of voids which lead to better aeration. By day 2 or 3, these conditions allow lactic acid bacteria to start out-competing the yeasts, eating up more of the sugar and creating a bunch of lactic acid while ethanol continues to build up.

The increase in ethanol coupled with increasing aeration allows acetic acid-producing bacteria to dominate by day 4. These guys are experts at breaking down ethanol into acetic acid, which causes the pH to plummet.

During all of those stages, an important physical change to the environment is happening. This fermentation box is a hotbed of metabolic activity, and, just like you and me (unless you're a cold, heartless bot, sorry @trufflepig), metabolism means heat production. For exactly the same reason that your backyard compost pile may feel hot and steams in morning, there's enough metabolic activity that the temperature of the box rises measurably and noticeably.

All of this heat, in combination with the low pH from all the acid, prevents the seeds from germinating and getting even more bitter. Instead, the heat and acid start penetrating into the seed and breaking them down into a bunch of flavor precursors.

Eventually, things get so hot and acidic that spore-forming aerobic bacteria take over. These bugs continue the process of biochemical conversion that leads to many compounds which, when heated, lead to the various flavors we perceive as 'chocolate'.

Compound(s)	Flavor
2-Methylpropanal, 2 (or 3)-methylbutanal	malty, dark chocolate
tetramethylpyrazine	nutty, coffee-mocha, roasted
3,5 (or 2)-diethyl-2 (or 5)-methylpyrazine	coffee, chocolate, rum, roasted
furaneol	caramel

A selection of interesting compounds produced during fermentation which lead to cocoa flavors. Adapted from here.

This is one of a few progressions, and is not inevitable. For example, if the box was more tightly sealed, the pulp would go anoxic and then anaerobic. There would be a lot of production of volatile fatty acids (which stink!), possibly hydrogen sulfide (which stinks like rotten eggs), and some methane production. This would make terrible chocolate.

If instead they let the fermentation continue, eventually molds like Geotrichum would take over, lending lots off 'off' flavors.

Milton Hershey's dubious microbial load

Apart from the necessary fermentation for chocolate production,there's an optional (and some would argue bad) fermentation which differentiates many classic American-style milk chocolates from European milk chocolate. I am a dark chocolate fan, myself, but milk chocolate is extremely popular and, as you certainly may have guessed from the name, milk is a vital ingredient for its production. Traditionally, chocolate factories in Europe were very close to their dairy supply. American factories, in comparison, could be days away from the nearest milk supply. Combine the long travel time with the lack of ubiquitous adequate, and reliable refrigerated transportation and you have a problem.

Milton Hershey's solution was essentially the same that many cultures have developed for preserving milk. Instead of trying to keep the milk from spoiling, you can instead allow it to go 'bad', but control the way it which it does so by controlling things like temperature, salinity, and the microbes present. This is how we get such wonderful things as cheese and yogurt. A key difference is that while most of these fermentations produce lactic acid, Hershey's process ends up producing butyric acid. I honestly do not know why they chose butyric, and the process is a trade secret. If I had to venture guesses, I would say that it's likely that the butyric-fermented milk still has some physical properties that are useful for making milk chocolate and that the production process involves optimized times and temperatures and massive dose of a starter culture.

The upshot of all this was that Hershey was able to produce a lot of milk chocolate cheaply and reliably. Beyond that, butyric acid has a distinctive flavor (in increasing amounts, it is what flavors parmigiano cheese, rotten butter, and vomit). If, like many US Americans, you grew up with it, this tastes natural and many other chocolate manufacturers actually just added butyric acid to serve the US palate. None of this was necessary in Europe, so US chocolate often tastes a little bit like puke to them. This is changing, with the upsurge in artisanal everything and increased international trade, a lot of chocolate in the US now does not have butyric acid in it. Conversely, I have to imagine there's at least one or two Europeans who, for whatever reason, prefer the Hershey's 'zing'.

Microbial succession. Not just for chocolate

Since microbial succession is so important for chocolate fermentation, it shouldn't be a surprise that people are trying to optimize the process using specific starter cultures and probably also controlling time, temperature, and aeration. This isn't just for chocolate though - microbial succession plays roles in everything - pond sediments, landfills, wastewater treatment, and other fermented foods are just a few examples. Understanding the processes which affect succession in these systems has some serious theoretical and practical implications.

Theoretical implications

When we talk about predicting the population of an ecosystem we often use the term community assembly. Boiling down a giant field of active research into two questions, community assembly is concerned with answering:

1. Given a starting population, an influx of immigrants, and environmental parameters, what community will we end up with?
2. If we want to arrive at a specific community composition, what starting population, etc should we begin with?

It turns out that answering those two questions is messy, complicated, and not straightforward at all. In fact, attempts to answer it have ended up developing whole new ways of talking about ecology which at first were in opposition to, then, more recently, integrated with the classic Darwinian niche-based selection processes we all grew up with. Understanding systems like chocolate fermentation can directly increase our ability to answer those big two questions.

Practical implications

Rebirth and renewal is of the major themes of Easter (and many of the other spring holidays which it is intimately related with). It is also arguably the major theme of the applied field of restoration ecology.

Oh, hi Ostara, goddess of Spring, whose symbols include bunnies and eggs! Source by Johannes Gehrts, in the public domain.

Basically, if an ecosystem is degraded, there is a whole branch of ecology which is concerned with restoring the "health, integrity, and sustainability" of that ecosystem. Imagine an abandoned industrial site that sat next to dredged marshes. If you wanted to restore that area to a robust, functional wetland, it would certainly help to have answers to the big two theoretical questions. Again, although a marshland involves many macrobiota, understanding (and even experimenting) with microbial model systems can help uncover basic ecological rules which lead to more effective restoration.

So, if you are expecting some chocolate soon, please, take the time to thank a microbe.

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†. I know I don't need to attribute CC0 stuff, but their stuff is gorgeous and I think you should check it out.

††. If you read up on the history of fermented food, a common theme is that someone, somewhere, somewhen left out a food source, it rotted, but they were too hungry (or possibly performing a prehistoric dare, have you seen a kombucha scoby?) to throw it out. We usually don't know this specifics of when or how this happened for any given fermented food, but dear ancestors, I salute you and your courageous stomachs.