Microbial Ecology of Food Production

Molecular methods to follow food and wine fermentations

Fermented Food and Beverage Production

Wine making illustrationFood fermentations are an amalgam of microbiological activities derived from both starter and non-starter microorganisms. The growth of non-starter (or indigenous) yeasts can significantly impact starter cultures and even inhibit normal starter function. Efforts in my laboratory have focused on molecular techniques that profile indigenous yeasts and bacteria in an effort to better understand the full range of microbial interactions that occur as well as track the impact of these interactions on the product quality.

Some questions about fermentation ecology that we are interested in:

  • Is there a "microbial terroir" associated with specific wineries and breweries....what about other fermented products like cheese, sake, coffee and chocolate?
  • What is the impact of metabolically active but non-culturable microbes in fermentation production systems?
  • How do microbes transfer within food and beverage production facilities?  Is there a seasonality to the microbial patterns observed>
  • Do patterns of microbial ecology on input food or beverage substrates (i.e. grape must, raw milk etc) impact the metabolite and flavor attributes of the final product.

Publications in this area (see a full list of Mills Lab publications here)

  • Bokulich, N. A., C. Masarweh, T. S. Collins, G. Allen, H. Heymann, S. E. Ebeler and D. A. Mills. 2016. Associations among wine grape microbiome, metabolome, and fermentation behavior suggest microbial contribution to regional wine characteristics. mBio 7:e00631-16.
  • Walker, G. A., A. Hjelmeland, N. A. Bokulich, D. A. Mills, S. E. Ebeler and L. F. Bisson. 2016.  Impact of the [GAR+] prion on fermentation progression and bacterial population composition with Saccharomyces cerevisiae strain UCD932.  American Journal of Enology and Viticulture. 67:296-307.
  • Dallas, D. C., F. Citerne, T. Tian, V. L. M.  Silva, K. M. Kalanetra, S. A. Frese, R. C. Robinson, D. A. Mills and D. Barile.  2016. Peptidomic analysis reveals proteolytic activity of kefir microorganisms on bovine milk proteins.  Food Chemistry 197: 273–284.
  • Bokulich, N. A., Z. T. Lewis, K. L Boundy-Mills, D. A. Mills.  2016.  A new perspective on microbial landscapes within food production. Current Opinion in Biotechnology. 37:182–189.
  • Swadener, M. L. and D. A. Mills. 2015. A new look at an old practice: How SO2 additions influence microbial diversity during fermentation. Wine & Viticulture Journal 3:32-34..
  • Zarraonaindia, I., S. M. Owens, K. West, J. Hampton-Marcell, S.Lax, N. A. Bokulich, D. A. Mills, G Martin, S. Taghavid, D. van der Lelie and J. A. Gilbert.  2015.  The soil microbiome influences grapevine-associated microbiota. mBio 6:e02527-14
  • Bokulich, N. A., J. Bergsveinson, B. Ziola, D. A. Mills. 2015. Mapping microbial ecosystems and spoilage-gene flow in brewery environments highlights patterns of contamination and resistance. Elife 4:e04634.
  • Porcellato, D., M. E. Johnson. K. Houck. S. B. Skeie. D. A. Mills. K. M. Kalanetra, J. L. Steele. 2015. Potential of Lactobacillus curvatus LFC1 to produce slits in Cheddar cheese. Food Microbiology 49: 65–73.
  • Bokulich, N. A., L. Amiranashvili, K. Chitchyan, N. Ghazanchyan, K. Darbinyan, N. Gagelidze, T. Sadunishvili, V. Goginyan, G. Kvesitadze, T. Torok and D. A. Mills. 2015. Microbial biogeography of the transnational fermented milk Matsoni. Food Microbiology46:121-131.
  • Bokulich, N. A., M. Swadener, K. Sakamoto, D. A. Mills and Linda F. Bisson.  2015. Sulphur dioxide treatment alters wine microbial diversity and fermentation progression in a dose-dependent fashion. American Journal of Enology and Viticulture. 66:73-79.
  • Ramsey, M. S., and D. A. Mills. Beer and Wine.   In, S. Doores, M.L. Tortorello, and B. Wilcke Eds, Compendium of Methods for the Microbiological Examination of Foods 5th Edition American Public Health Association, Washington DC, (In Press).
  • Bokulich, N. A., M. Ohta, M. Lee and D. A. Mills. 2014. Indigenous bacteria and fungi drive traditional kimoto sake fermentations. Applied and Environmental Microbiology 80:5522-5529.
  • Bokulich, N. A., J. H. Thorngate, P. M. Richardson and D. A. Mills. 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 D. A. Mills.  2013. Improved internal transcribed spacer primer selection enables quantitative, ultra-high-throughput fungal community profiling.  Applied and Environmental Microbiology 79:2519-2526.
  • Ramsey, M. S. and D. A. Mills.   2013.  Winery Biofilms:  A source for contamination throughout the winery.  Practical Winery and Vineyard Journal.  Winter Issue.
  • Bokulich, N. A., S. Subramanian, J. J. Faith, D. Gevers, J. I. Gordon, R. Knight, D. A. Mills, J. G. Caporaso.  2013. Quality-filtering vastly improves diversity estimates from Illumina amplicon sequencing. Nature Methods 10:57-59.
  • 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.
  • Bokulich, N. A., C. W. Bamforth and D. A. Mills. 2012. A review of molecular methods for microbial community profiling of beer and wine.  Journal of the American Society for Brewing Chemists 70:150-162.
  • Bokulich, N. A., C. M. L. Joseph, G. Allen, A. K. Benson and D. A. Mills. 2012. Next-generation sequencing reveals significant bacterial diversity of botrytized wine. PLoS One 7:e36357.
  • Bokulich, N. A., C. Bamforth, and D. A. Mills. 2012. Brewhouse-resident microbiota responsible for fermentation of American coolship ale. PLoS One 7:e35507.
  • Bokulich, N. A. and D. A. Mills.  2012.  Differentiation of mixed lactic acid bacteria communities in beverage fermentations using targeted terminal restriction fragment length polymorphism.  Food Microbiology 31:126-131.
  • Bokulich, N., C. H. Hwang, L. Shuwen, K. Boundy-Mills and D. A. Mills.  2012. Profiling the yeast ecology of wine using terminal restriction fragment length polymorphism analysis.  American Journal of Enology and Viticulture 63:185-194. (AJEV best Paper award).
  • Marcobal, A. and D. A. Mills. 2009.  Genomics of Oenococcus oeni and other lactic acid bacteria. In Koning, H., Uden, G. and Frohlich, J. Eds. Biology of Microorganisms on Grapes, in Musts and Wine. Springer, New York.
  • Marcobal, A. D. A. Sela, K. Makarova, Y. Wolf and D. A. Mills.  2008. Hypermutability in Oenococcus. Journal of Bacteriology. 190:564-570.  (Cover article).
  • Mills, D. A., T. G. Phister, E. Neeley and E. Johannsen.  2007. Wine Fermentations.  In Molecular Methods and Microbial Ecology of Fermented Foods.  Springer, New York.
  • Scharfen, E. C., D. A. Mills and E. A. Maga. 2007. Utilization of human lysozyme transgenic goat milk in cheese-making: Effects on lactic acid bacteria profile.  Journal of Dairy Science. 90:4084-4091.
  • Phister, T. G., H. Rawsthorne, L. Joseph and D. A. Mills. 2007. A quantitative real-time PCR assay for detection and enumeration of Hanseniaspora sp. from wine and juice. American Journal of Enology and Viticulture 58: 229-233.
  • Makarova, K., A. Slesarev, Y. Wolf, A. Sorokin, B. Mirkin, E. Koonin, A. Pavlov, N. Pavlova, V. Karamychev, N. Polouchine, V. Shakhova, I. Grigoriev, Y. Lou, D. Rohksar, S. Lucas, K. Huang, D. M. Goodstein, T. Hawkins, V. Plengvidhya, D. Welker, J. Hughes, Y. Goh, A. Benson, K. Baldwin, J.-H. Lee, I. Díaz-Muñiz, B. Dosti, V. Smeianov, W. Wechter, R. Barabote, G. Lorca, E. Altermann, R. Barrangou, B. Ganesan, Y. Xie, H. Rawsthorne, D. Tamir, C. Parker, L. McKay, F. Breidt, J. Broadbent, R. Hutkins, D. O’Sullivan, J. Steele, G. Unlu, M. Saier, T. Klaenhammer, P. Richardson, S. Kozyavkin, B. Weimer, and D. A. Mills. 2006. Comparative genomics of the lactic acid bacteria. 2006. Proceedings of the National Academy of Sciences. 103:15611-15616.
  • Mills, D. A. and E. Neeley. 2006. Molecular methods to characterize wine microorganisms. International Wine Microbiology Symposium Proceedings, Yosemite, CA, CATI. P. 77-84.
  • Neeley, E., T. Phister, and D. A. Mills. 2005. A differential real-time PCR assay for enumeration of lactic acid bacteria in wine. Applied and Environmental Microbiology. 71:8954-8957.
  • Mills, D. A., H. Rawsthorne, C. Parker, D. Tamir, and K. Makarova. 2005. Genomic analysis of Oenococcus oeni PSU-1 and its relevance to winemaking.  FEMS Microbiology Reviews. 29: 465-475.
  • Mills, D. A.  2004.  Practical Implications of the lactic acid bacteria genome project:  Malolactic fermentations.  Proceedings of the XVIes Entretiens Scientifiques Lallemand.  pg 39-43.
  • Mills, D. A. 2004. The lactic acid bacteria genome project. Journal of Food Science, 69: FMS28-30.
  • Phister, T. and D. A. Mills.  2003. Real-time PCR assay for detection and enumeration of Dekkera bruxellensis in wine. Applied and Environmental Microbiology. 69:7430-7434.
  • Lopez, F. Ruiz-Larrea, L. Cocolin, E. Orr, T. Phister, M. Marshall, J. VanderGheynst and D. A. Mills. 2003. Design and evaluation of PCR primers for direct analysis of bacterial populations in wine.  Applied and Environmental Microbiology. 69:6801-6807
  • Marshall, M. N., L. Cocolin, D. A. Mills and J. S. VanderGheynst.  2003. Evaluation of PCR primers for denaturing gradient gel electrophoresis analysis of fungal communities in compost.  Journal of Applied Microbiology.  95:934-948.
  • Cocolin, L. and D. A. Mills. 2003. Wine yeast inhibition by sulfur dioxide: a comparison of culture-dependent and –independent methods. American Journal of Enology and Viticulture. 54:125-130
  • Mills, D. A., E. Johannsen, and L. Cocolin. 2002. Yeast diversity and persistence in botrytis-affected wine fermentations. Applied and Environmental Microbiology 68:4884-4893
  • Klaenhammer, T, E. Altermann, F. Arigoni, A. Bolotin, F. Breidt, J. Broadbent, R. Cano, S. Chaillou, J. Deutscher, M. Gasson, M. van de Guchte, J. Guzzo, T. Hawkins, P. Hols, R. Hutkins, M. Kleerebezem, J. Kok, O. Kuipers, M. Lubbers, E. Maguin, L. McKay, D. A. Mills, A. Nauta, R. Overbeek, H. Pel, D. Pridmore, M. Saier, D. van Sinderen, A. Sorokin, J. Steele, D. O'Sullivan, W. de Vos, B. Weimer, M. Zagorec, and R. Siezen. Discovering lactic acid bacteria by genomics. 2002. Antonie van Leeuwenhoek 82:29-58.
  • Cocolin, L., A. Heisy and D. A. Mills. 2001. Direct identification of the indigenous yeasts in commercial wine fermentations. American Journal of Enology and Viticulture. 52:49-53.
  • Cocolin, L., L. F. Bisson, and D. A. Mills. 2000. Direct profiling of the yeast dynamics in wine fermentations. FEMS Microbiology Letters 189:81-87.


This work has received funding from:

  • The Sloan Foundation
  • Constellation Brands
  • American Vineyard Foundation
  • California Competitive Grants Program for Research in Viticulture and Enology