Progress 01/12/07 to 09/30/12
OUTPUTS: todate we have published 11 papers with 4 more in preparation and presented 18 posters or papers PARTICIPANTS: Mara Massel- technical Michael Lenordelii M.S. student Victoria Gray M. S. Student Megan Beckner M.S. student Jinjun Gong-Postdoc Steven Gray-Postdoc Melissa Ivey M. S. Student Vanessa Pittet-(visiting PhD Student) Blake Layfield M.S. student TARGET AUDIENCES: In this project we developed knowledge that would help fermented beverage and biofuel producers prevent or control contaminating organsims. We developed at least one assay to detect spoilage microbes and explored the genomes and physiology of dekkera and pediococcus both major contaminants in biofuel fermentations. these studies will lead to better control systems for these spoilage agents. In addition, we trained nine researchers two of which have gone onto work in the food fermentation industry. In addition two other M.S. students ahve gone onto PhD programs still focused in the fermented foods industries. The first Megan Beckner is doing a PhD working on wine in Italy. Blake is working on Brewing at NCSU. Finally Michael Leanordelli is working as an Extension Associate at the University of Missouri. Both postdocs have gone into the biofuel industry. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
We have sequenced the genome of the major wine spoilage agent Dekkera bruxellensis which will provide insight into the physiology of this spoilage organism. We have developed assays to detect Dekkera anomola, looked at gene expression for the fermentation contaminating microbe Pediococcus claussenii and determined that flavor compounds produced by lactobacilli can impact Saccharomyces during the course of a fermentation.
- Gray, Victoria, and T. G. Phister. 2011. The influence of yeasts in the survival of probiotics in yogurt. IFT. New Orleans, LA. (June 2011)-poster
- Beckner, Megan, and T. G. Phister. 2011. The effects of aromatic alcohols on the growth of Saccharomyces cerevisiae during fermentation. ASM. New Orleans, LA. (May 2011)-poster
- Phister, Trevor G. 2011. Cell-cell signaling in microbial interactions. Plant pathology Society of North Carolina, Raleigh, NC. (March 2, 2011)-presentation
- Gray, Steven R., H. Rawsthorne, B. Dirks and T. G. Phister. 2011. Detection and Enumeration of Dekkera anomala in Beer and Cola Using Real-Time PCR. Lett. Appl. Microbiol. 52:352-359
- Rawsthorne, H., T. G. Phister and L. Jaykus. 2009. Development of a fluorescent in situ method for visualization of enteric viruses. Appl. Environ. Microbiol. 75:7822-7
- Rawsthorne, H. and T. G. Phister. 2009. The presence of Saccharomyces cerevisiae DNA in various media used to propagate yeasts and its removal by ethidium monoazide bromide. Lett. Appl. Microbiol. 49: 652-654
- Phister, T. G. 2009. Using Microbial Succession to the Processor's Advantage: Food Fermentation and Biocontrol. In L. Jaykus, H. Wang, and L. Schlesinger (ed.), Foodborne Microbes: Shaping the Host Ecosystem. ASM Press, Washington D. C.
- Gray, Steven, J. Gong, John Sheppard and Trevor G. Phister. 2010. Bioethanol Production by the yeast Kluyveromyces marxianus using five and six carbon sugars. ASM, San Diego, CA. (May 23-27, 2010)-poster
- Phister, Trevor G. 2010. Enhancing Bioethanol Fermentation using Kluyveromyces marxianus. Novozymes, Franklinton NC (July 9, 2010)-Presentation
- Corro-Herrera, V. A., Phister, T., Mendoza-Garcia, P., and Aguilar-Uscanga, M. G. 2010. Acetic acid production byBrettanomyces bruxellensis in continuous culture. Fourth International Congress on Food Science and food Biotechnology in Developing Countries. Veracruz, Mexico
- Beckner, Megan, Melissa Ivey, Mara Massel and Trevor G. Phister. 2010. Interactions between Saccharomyces cerevisiae and Lactobacillus species during fermentation. 110th IFT Annual Meeting. Chicago IL. (July 17-20, 2010)-Poster
- Gray, Victoria, Michael Leonardelli and Trevor G. Phister. 2010. Affect of Saccharomyces on the growth of Dekkera bruxellensis. 110th IFT Annual Meeting. Chicago, IL. (July 17-20, 2010)-Poster
- Massel, Mara and Trevor G. Phister. Affect of amplicon size on the differentiation of viable and nonviable Dekkera bruxellensis using EMA and real-time PCR. 2010. Society for Industrial Microbiology Annual Meeting. San Francisco, CA. (August 1-5, 2010)-Poster
- Trevor Phister. 2009. Functional genomic analysis of Saccharomyces-Lactobacilli Interactions in Bio-ethanol fermentations Genes to Products Agricultural Plant, Microbe, and Biobased Product Research. USDA Bethesda MD
- Aguilar-Uscanga, U. and T. G. Phister. 2008. Use of propidium monoazide (PMA) for live/dead distinction of Brettanomyces bruxellensis. 108th General Meeting American Society for Microbiology. Boston
- H. Rawsthorne, T. G. Phister and L.A. Jaykus. 2008. Fluorescent labeling of mouse norovirus facilitates the study of interactions between enteric viruses and foods. 108th General Meeting American Society for Microbiology. Boston
- J. Blake Layfield, Trevor G. Phister and John Sheppard. 2009. Characterization of hybrid strains of Saccharomyces pastorianus as related to desiccation tolerance and fermentation performance. ASBC, Tucson AZ
- Piskur, J. Z. Ling, M. Marcet-Houben, O. P. Ishchuk, A. Aerts, K. LaButti, A. Copeland, E. Lindquist, K. Barry, C. Compagno, L. Bisson, I. V. Grigoriev, T. Gabaldon and T. Phister. 2012. The genome of wine yeast Dekkera bruxellensis provides a tool to explore its food-related properties. Int. J. Food Microbial. 157: 202-209
- Ivey, M. and T. G. Phister. 2011. Detection of Common Microorganisms in Wine: A Review of Molecular Techniques. J. Industrial Microbiol Biotech. 38: 1619-1634
- Beckner, M., M. Ivey, and T. G. Phister. 2011.Contamination in Biofuel Fermentations. Lett Appl. Microbiol. 53: 387-394
- Uscanga, M. G. A., Y. G. Alvarado, T. G. Phister, M. L. Delia and P. Strehaiano. 2011. Modeling the growth and ethanol production of Brettanomyces bruxellensis at different glucose concentrations. Lett Appl. Microbiol. 53:141-149
- Layfield, J. B., T. G. Phister and J. D. Sheppard. 2011. Characterization of Hybrid Strains of Saccharomyces pastorianus for Desiccation Tolerance. J. Am. Soc. Brewing Chem. 69:108-115
- Rawsthorne, H. and T. G. Phister. 2009. Detection of viable Zygosaccharomyces bailii in fruit juices using ethidium monoazide bromide and real-time PCR. Int. J. Food Microbiol. 131:246-250
- Pettit, Vanessa, T. G. Phister and Barry Ziola. 2012. Pediococcus claussenii genetic expression during growth in beer assessed by transcriptome sequencing (RNA-seq). World Brewing Congress, Portland OR, USA. (July 28-August 1, 2012)-presentation
- Pettit, Vanessa, Mara Massel, Barry Ziola and T. G. Phister. 2011.RNA-seq of Pediococcus claussenii during growth in beer. 10th Symposium on Lactic Acid Bacteria. Egmond aan Zee, Netherlands. (August28-September1, 2011)-presentation
- Gray,S., L. Bisson, J. Piskur, F. Detrich, T. Henick-Kling, S. Baker and T. G. Phister. 2010 Completion of the Dekkera(Brettanomyces) bruxellensis Genome Sequence JGI User Meeting, Walnut Creek, CA
- Phister, T. 2009. Functional genomic analysis of Saccharomyces-Lactobacilli Interactions in Bio-ethanol fermentations 95th Annual ASM Southeastern Branch Conference. Savannah, GA
- Jinjun Gong, John Sheppard, and Trevor G. Phister. 2009. Characterization of a newly discovered Kluyveromyces marxianus strain for its carbohydrate utilization and ethanol production compared to commercial Saccharomyces cerevisiae strains CAT-1 and SuperstartTM. the 27th International Specialized Symposium on Yeast. Paris
- Melissa, Ivey, Mara Massel and Trevor G. Phister. 2009. Effects of lactic acid bacteria on ethanolic fermentations of Saccharomyces cerevisiae the 27th International Specialized Symposium on Yeast. Paris, poster
- Maria Guadalupe Aguilar-Uscanga, Mara Massel and Trevor G. Phister. 2009. The effect of ethanol and sulfite on the growth of Brettanomyces. XII National congress of Biotechnology and Bioengineering and VII International symposium of alcohol and yeast production. Acapulco Mexico
Progress 10/01/07 to 09/30/08
OUTPUTS: We optimized the DNA isolation procedures comparing the use of a kit with boiling. Samples were prepared by boiling and gave a time to fluorescence for 100 cells of 15.12 minutes using LAMP-PCR,staining with sybergreen and detection on a multiwell plate reader. The kit gave a time to fluorescence of 12.7 minutes. Thus the boiling method worked as well as the kit at a much lower cost and time commitment. To determine the best visualization method we first needed to determine the limit of detection for the assay. Using a series of 1 in ten dilutions DNA was isolated from 10 million cells down to 1 cell. Using either extraction method we detected Brettanomyces down to 10 cells when the samples were visualized by gel electrophoresis. We next attempted to apply the use of sybergreen staining and a fluorometer to detect the DNA amplification. Using this method we were able to detect 10 cells. We also detected the fluorescent color change by the naked eye. Using this method we were able to detect down to 100 cells. We are currently trying to optimize this detection method through use of other dyes different reaction volumes and addition of handheld UV lights. We are currently adapting the assay for enumeration of the population in wine. We diluted Brettanomyces in media, wine, and wine plus Saccharomyces. The DNA was isolated by both methods and subjected to the LAMP-PCR assay. To enumerate the yeast we monitored the time to a predetermined fluorescence reading and plotted this for cultures of known population sizes. This generates a standard curve of time versus cell number. Using this method, we can now generate standard curves down to 10 yeasts. Additionally we worked on the detection of viable Brettanomyces using Propridium monoazide (PMA) and real-time PCR. PMA is a dye that is excluded from living cells but can penetrate the cell membranes of dead cells. Once inside it binds to the organisms DNA and blocks amplification by PCR. The goal of this work was to examine the suitability of PMA to selectively remove genomic DNA of dead cells from yeast culture. Incubation time PMA concentration and light exposure were all optimized. These conditions generated a strong correlation (r2=0.994) between CT and CFU/mL of viable cells. To elucidate the relationship between the proportion of viable cells, the DNA yield and the QPCR signals after PMA treatment, mixtures with defined ratios of viable and dead cells were used. An aliquot of B. bruxellensis was subjected to heat treatment resulting in a decrease in culturable cell counts to zero. Heat-killed cells were mixed with the untreated original culture in defined ratios with viable cells representing 0,10,20,30,40 and 50% of the total respectively. Increasing proportions of viable cells led to a substantial increase in the amount of genomic DNA not bound by PMA and to decreasing CT values in QPCR. While using only heat killed cells presented the highest CT value, i.e. greatest signal reduction. A plot of percent viability versus CT values revealed a linear correlation with a R2 value of 0.997. This suggested that the PMA-QPCR assay quantified only the living Brettanomyces cells. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
As this is the first year of the project there is limited impact from the results. We are however hoping that the LAMP assay will be developed and useful for winemakers as an inexpensive method to test for the spoilage yeast Brettanomyces. The PMA assay will also provide a future impact if it is able to develop a better correlation between the Brettanomyces population present in wine and the production of spoilage compounds.
- M. G. Aguilar-Uscanga and T. G. Phister. Use of propidium monoazide (PMA) for live/dead distinction of Brettanomyces bruxellensis. 108th General Meeting American Society for Microbiology. Boston (June 2008)-poster