In order to see the role of fungi in chocolate, we need to know a little about how chocolate is produced. The "chocolate tree," Theobroma cacao grows in humid areas of the rainforest. In their native habitat they grow tall and straight (up to 100 ft) to try to outcompete the competition, but in plantations they grow much shorter and more branched. Like most trees in the rainforest, cacao is evergreen, losing its leaves only gradually during the year while growing new ones. It is during their growth that the first fungus is needed. Several species of fungi form mycorrhizae with the roots of the cacao tree. Remember that the mycorrhiza is a mutualistic relationship, and thus both partners benefit. In the case of cacao, these are endomycorrhizal fungi. Unlike ectomycorrhiza species such as Boletus edulis, chanterelles, truffles, the dog turd fungus, milk mushrooms, and many others, endomycorrhizal fungi do not form macroscopic mushrooms. In other words, their fruiting bodies are normally invisible to the naked eye, although some of the larger ones can reach just over 1 mm in diameter. If you read the information on the links above, you will remember that ECTOmycorrhizal fungi form a sheath, called a mantle, that surrounds the roots. Exchange of nutrients between the partners is through a Hartig net, hyphae that penetrate between the cells of the plant root cortex. ENDOmycorrhizal fungi, on the other hand, do not form a mantle. More importantly, exchange of nutrients takes place with arbuscules, highly branched hyphal modifications that penetrate into the cells of the cortex. Both types of mycorrhizae put hyphae out into the soil to scavenge for water and nutrients, thus benefiting the plant while they receive sugars from the plant's photosynthesis.

As it turns out there are many species of fungi that form endomycorrhizae with cacao. The most common one in Venezuela (and the one on which I can find the most information) is Acaulospora scrobiculata. Some species of Gigaspora, Scutellospora, and Glomus, particularly G. etunicatus, also seem to form endomycorrhizae with cacao. In general endomycorrhizal relationships tend to be more "promiscuous" than ectomycorrhizae. There are relatively few species of endomycorrhizal fungi identified, and they have a wide host range. Similarly, endomycorrhizal plants can form relationships with a wide range of fungi.

All of these endomycorrhizae formers are microscopic "lower" fungi. Most taxonomic schemes accept these genera as members of the phylum Glomeromycota formerly called the order Glomales (Glomerales) in the Zygomycota. Recent evidence indicates that these endomycorrhizal fungi should be placed in their own new phylum, the Glomeromycota. The molecular evidence for this new phylum is very compelling, and certainly these fungi are quite different from any of the other four phyla.

In cacao, the pink and white flowers grow in clusters directly on the trunk. This is called "cauliflory" and is likely a special adaptation to allow for better support of the rather heavy fruit. After pollination by small mosquito-like midge flies, the fruits, called pods, develop from the ovaries of the flowers on the sides of the trunk. A mature tree can produce twenty to fifty pods per year, with more than 50 seeds per pod. It is from these seeds that chocolate is made, and it takes about one pod of seeds to make a 100g chocolate bar (about 4 ounces). At this point the beans themselves are very bitter, and no animal is interested in eating them. However, the seed coat is sweet, white, and pulpy; animals such as birds and monkeys (or graduate students) are enticed to eat the pulp and discard the bitter seeds, effectively dispersing the seeds throughout the forest. If you think about this, it makes sense that the seeds should be bitter to avoid being eaten.

So how did humans find out how to eat these bitter seeds? History in unclear on this, but it is likely that native peoples tried some of the rotten seeds and found them to be delicious. Eventually it was discovered that the beans must be fermented. In the sense of food scientists, fermentation means that the beans must be partially decayed, removing the bitter taste and breaking down the beans into deliciousness. Fermentation takes place with two fungi, Candida krusei and Geotrichum. Candida is a true yeast, with a single celled vegetative body, while Geotrichum is yeast-like, with hyphae that break up into yeast. Most of the time these fungi are already present on the cacao pods and seeds, so, if left to their own means, the fermentation will take place. However in modern chocolate making, specific strains of the two fungi are added. These are strains that have been selected over hundreds of years for providing optimum flavor and aroma to the chocolate. Each chocalate company has its own specific proprietary strains that they guard from other companies. Let's consider these two fungi separately.

Candida krusei (shown to the left) is a budding yeast in the same genus as Candida albicans, the major cause of yeast infection in humans. However, C. krusei only very rarely causes human infection-- and live yeasts are not found in chocolate. Like most other yeasts (such as Saccharomyces cerevisiae) Candida replicates by budding. This means that the single-celled yeast blebs off a much smaller daughter cell, usually more than one. Each daughter cell eventually falls off and later produces its own daughter cells, thus propagating the species. the yeasts can divide once every couple hours, and soon there are thousands of yeasts in a small area, producing their enzymes to break down the pulp on the outside of the beans. This makes acetic acid, which kills the embryo of the seed and develops the chocolately aroma, in the process eliminating the bitterness in the beans.

The other fungus that has a role in fermentation of cacao into chocolate is Geotrichum (shown to the right). This fungus can grow as a mycelium, composed of microscopic threads called hyphae. However, these hyphae can break up into small squares called arthroconidia-- just a fancy name for square yeasts. These arthroconidia typically enlarge, their corners round off, and they eventually look like typical yeasts under growing conditions. Under these conditions, Geotrichum produces a different set of enzymes that break down the pulp, release acetic acid, and gets rid of some of the other bitter components in the beans. Interestingly, from a taxonomic point of view, it is unclear which species of Geotrichum is found here. No scientists seem to want to commit to naming which species it is! To the right is Geotrichum candidum, a common species in North America.

After fermentation, the waste hulls are removed, often being sold as mulch for the garden. Next the beans are ground into a fine powder, as Becky Curland demonstrates to the left. In Costa Rica, they still often grind the beans with the same kinds of stones as the Aztecs and Mayans did those many years ago. Sugar is added, the mixture is stirred vigorously with special tools until it becomes foamy, and there we have hot chocolate. This is the form in which chocolate became famous, becoming important especially in long sea voyages. It was easy to store as a dry powder, and it was certainly easy enough to boil water. It's strange to think of it now, but hot chocolate was at first made with just water by the Mayans and the Aztecs, but more recently milk has become a more popular alternative. Surprisingly the first record of milk being used to make hot chocolate was 1727.

Many years later, people found a way to more easily transport chocolate-- the chocolate bar! These are made in a similar way to the cocoa powder, but it is usually ground more finely. Cocoa butter is removed to make a dry powder. This dry powder is mixed with sugar and part of the cocoa butter at high temperature for up to 24 hours in a process called conching. The product must then be cooled slowly to maintain the integrity of the bar; that is preventing the cocoa butter from leaching out of the mixture. While still warm, the chocolate is poured into molds (the metal ones not the fungal kind) and allowed to cool into its final shape.

Of course there are many variations in the specifics of the steps in chocolate making. Each company has its own proprietary methods, including what kind of beans are used, how the beans are roasted, how much cocoa butter is added back to the chocolate and the specific conditions in the final steps. That's why all chocolate tastes different. Of course, some companies even add wax as kind of a "chocolate helper." It's better to get the good stuff.

All that being said, I'm one of those weirdoes who doesn't really like chocolate. I'm was the one in kindergarten who wanted white milk instead of chocolate milk. I finally gave into peer pressure and started drinking chocolate milk, even though I didn't really like it. Yuk. However, there are a few kinds of chocolates I like, especially a good Swiss dark chocolate. Most of the rest you are welcome to have. The worst thing you could make me eat to torture me would be a Devil's food cake with chocolate frosting covered with chocolate ice cream. I usually lie and say I'm allergic to chocolate so as not to offend the host.

I hope you enjoyed learning about chocolate and the fungi that are necessary for you to enjoy that sweet delicious treat. Be sure to think about fungi the next time you have chocolate, or when you get that box of chocolates from that special someone on Valentine's day.

References

• Cuenca, Gisela and Erasmos Meneses. 1996. Diversity patterns of arbuscular mycorrhizal fungi associated with cacao in Venezuela. Plant and Soil 183:315-322.
• International Collection of (Vesicular) Arbuscular mycorrhizal Fungi.
• West, John A. 1992 "A brief history and botany of Cacao." in Chilies to chocolate: foods the Americas gave to the world. Nelson Foster and Linda Cordell, eds. Tucson: University of Arizona Press

If you have anything to add, or if you have corrections, comments, or recommendations for future FotM's (or maybe you'd like to be co-author of a FotM?), please write to me at volk.thom@uwlax.edu

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