08/08/14 | Posted in profiles by Benjamin Wolfe

During his PhD research in the Mills Lab at the University of California Davis, Nicholas (Nick) Bokulich completely transformed how we view the microbial diversity of many fermented foods in the US. From wine to cheese to sake, Nick’s research opened up new dimensions of the microbial diversity of these traditional foods. In this Profile, Nick talks about improvements in DNA-sequencing technology, terroir, and the future of food microbiology.

Your Ph.D. research at UC Davis included some of the first studies of traditional fermented foods being produced in the United States. Did you find anything unique about the microbes in the US compared to similar foods produced/aged in Europe or other parts of the world?
It is tough to say that we discovered unique microbial signatures in these foods relative to their European equivalents without doing a side-by-side comparison and, unfortunately, we did not get our hands on any equivalent European fermentations. Most prior studies of fermented foods (e.g., cheese, beer, and wine), used culture-based or older molecular techniques (e.g., DGGE, TRFLP). Using next-generation sequencing, we see a much richer microbial profile in these foods than observed previously. For example, in botrytized wines we detected species of Methylobacterium and Sphingomonas, two genera that had never been detected in wines previously to our knowledge. Acting on this information, we were able to selectively culture these organisms from finished wines. We are seeing these and other organisms in more and more US wines as we sequence, so it is unclear whether these are unique to US wines or whether they simply haven’t been detected in earlier studies of European wines. Until these same modern methods are used to update our knowledge of the microbiota in the same European fermentations, we can’t truly answer this question!

Based on what we do know, however, most of the organisms that are primarily responsible for European fermentations are also seen in the US. For example, Saccharomyces cerevisiae and some other non-Saccharomyces yeasts (Hanseniaspora, Candida zemplinina, etc) and bacteria (especially Oenococcus) are primarily responsible for wine fermentations in Europe and the US. For another example, S. cerevisiae and Brettanomyces species are the main drivers of both Belgian lambic fermentations and American coolship ale fermentations; however, we do see different bacterial profiles in these beers which may explain some of their sensory differences. In US food fermentations, however, we frequently see regional microbial patterns, such as in grape musts from different growing regions of California. If regional conditions are shaping food fermentation microbiota even within a single state of the union, it seems reasonable to expect the same regional differences to apply over a larger scale, e.g., between the equivalent products in Europe and globally. It also depends on the food, and human processing conditions play a large role in shaping the microbiota of the fermentation itself, as opposed to the raw ingredients.

How have new DNA sequencing technologies changed our view of the microbial communities that make fermented foods?
These methods are extremely sensitive and very high-throughput, which means that we can more comprehensively characterize microbial communities and test many more samples than was possible with earlier methods (e.g., culture-based methods and older molecular methods like DGGE). Due to this higher sensitivity, we are seeing richer microbial communities than previously described in these foods, for example the Sphingomonas and Methylobacterium in wines that I mentioned above. Due to the high-throughput nature, we can examine so many more samples simultaneously, which allows us to address much larger and complex questions pertaining to the production of these foods. For example, we can map out the microbial profiles of complete production facilities over space and time, increasing our understanding of how microbes are transmitted in these facilities and identifying spoilage reservoirs. We can compare fermentations from many different locations and at many different stages, solving questions about how regional factors shape the microbial communities of food fermentations and the biogeographical factors underlying these phenomena. These tools equip us to answer questions about which we could only have dreamt previously.

What will the future of food microbiology look like? Where is the field headed and how will new discoveries in food microbiology impact the products available to consumers?
Current research is elaborating the inextricable role of microbes in every aspect of food production. This will lead to greater acceptance and appreciation of microbial activity in foods as producers increasingly choose to embrace these many roles with an open mind. We are learning about how regional factors, human practices, processing conditions, and many other external factors interact with the microbiota of foods.This leads to a two-fold path: a better understanding of how to control and manipulate this complex consortium but also a better understanding of what we need to leave untouched to preserve the good foods we have already.

Many people (perhaps justifiably) see research like mine and think that the goal is automatically to hijack terroir for corporate good. My goals are to improve the way foods are grown and processed, with an eye to preservation. By understanding how regional climatic factors shape the microbiota of grape surfaces, we can match appropriate grape varieties to appropriate growing regions, so that growers and winemakers large and small can make the right planting choices, and select a more diverse range of grape varieties to fit the variegated landscape, rather than carpeting the planet with Chardonnay.

What are some of the greatest microbiological challenges faced by artisan fermented food producers in the US?
Contamination and consumer acceptance. The problems facing artisan producers in the US are not actually that different from artisan producers worldwide, or even from those faced by industrial producers. Microbial contamination is an ever-present threat to producers big and small, as any unintended microbial activity that influences the sensory properties of the food (aroma, flavor, texture, color, etc.) can be considered spoilage. Traditional processes are usually what make these products so prized by consumers, but are also what make them more susceptible to microbial contamination from raw ingredients, humans, and the environment. We need to better understand how microbial communities are established and maintained in artisan food processing facilities just as much as at industrial processing plants. This will allow us to understand how these microbes interact with the foods during processing, and to identify indoor conditions, materials, processes, and activities that promote or suppress the growth and transmission of contaminants in the processing environment.

Consumer acceptance comes into play when you consider that one person’s contaminant is another person’s beneficial microbe. The microbes that make Taleggio taste tremendous would not sit so well on a Camembert. Historically and traditionally, it was the regional conditions that defined how different foods were made (i.e., processing techniques) and the conditions that they encountered both pre- and post-processing, thereby shaping the microbial communities of these foods and their activities. Thus, whatever environmental microbes were selected for growth in/on these foods came to define the sensory properties of these foods. This was of course also driven by consumer preference centuries ago just as it is now, but to a certain degree consumers were also trained to accept and expect certain foods to look, taste, and smell specific ways. Hence our expectations of Taleggios and Camemberts today has been shaped by historical trends. US artisanal producers need to walk a fine line between emulating traditional styles and pushing the envelope in defining the unique character of their own foods — all while working under local constraints (microbiologically, technologically, climatically, etc).

From a microbiological standpoint, it bears consideration that undesirable microbial activity (e.g., some pink mold growing on a cheese rind or wild yeast activity in a wine fermentation) may simply be the local terroir making itself known, though from a consumer acceptance standpoint this might commonly be considered contamination –  a cheesemaker may have a difficult time trying to sell a neon pink wheel of gorgonzola, no matter how good the cheese inside tastes. Finding the right balance between consumer acceptance and microbial constraints is a key challenge…. as is gradually opening up consumers’ minds to trying new, unusual varieties that express American terroirs (microbial and otherwise). This challenge is borne equally by cheesemakers, winemakers, brewers, kombucha makers, etc.

What is your favorite food microbe?
I hate to give the most trite and unexciting answer, but I must say Saccharomyces cerevisiae. It is like saying your favorite type of ice cream is chocolate, but I just honestly love the little critters. This single yeast species is responsible for more food fermentations than any other (except for lactic acid bacteria) and they just happen to be my favorites. Beer, wine, breads, sake, whisky… I also happen to rather like chocolate ice cream.

Nick is currently doing post-doctoral research in Dr. Martin Blaser’s lab at New York University. He’s also the co-founder and technical director of MicroTrek, Inc., a food research and service company providing microbiota monitoring for the food and beverage industry.

Interview conducted and edited by Benjamin Wolfe. Center header photo courtesy of Nicholas Bokulich. Right data panel in header photo is from this PLoS One paper on seasonal changes in wine microbes by Bokulich et al.

Check out some of Nick’s research papers (this is just a selection – there are many more!) on the microbial ecology of food and fermentations:

Bokulich, N. A. et al. 2014. Microbial biogeography of wine grapes is conditioned by cultivar, vintage, and climate. Proceedings of the National Academy of Sciences 111:E139-148.

Bokulich, N. A. and D. A. Mills. 2013. House microbiome drives microbial landscapes of artisan cheesemaking plants. Applied and Environmental Microbiology 79:5214-5223.

Bokulich, N. A., M. Ohta, P. M. Richardson and D. A. Mills. 2013. Monitoring seasonal changes in winery-resident microbiota. PLoS One 8:e66437.

Bokulich, N.A. and Bamforth, C.W. 2013. The microbiology of malting and brewing. Microbiology and Molecular Biology Reviews 77:157-172.

Bokulich, N. A. et al. 2012. Brewhouse-resident microbiota responsible for fermentation of American coolship ale. PLoS One. 7(4): e35507.

Bokulich, N. A. et al. 2012. Next-generation sequencing reveals significant bacterial diversity of botrytized wine. PLoS One. 7(5): e36357

Bokulich, N, and D. A. Mills. 2012. Next-generation approaches to the microbial ecology of food fermentations. Journal of Biochemistry and Molecular Biology 45:377-389.