Next-Generation Sequencing Reveals Significant Bacterial Diversity of Botrytized Wine

While wine fermentation has long been known to involve complex microbial communities, the composition and role of bacteria other than a select set of lactic acid bacteria (LAB) has often been assumed either negligible or detrimental. This study served as a pilot study for using barcoded amplicon next-generation sequencing to profile bacterial community structure in wines and grape musts, comparing the taxonomic depth achieved by sequencing two different domains of prokaryotic 16S rDNA (V4 and V5). This study was designed to serve two goals: 1) to empirically determine the most taxonomically informative 16S rDNA target region for barcoded amplicon sequencing of wine, comparing V4 and V5 domains of bacterial 16S rDNA to terminal restriction fragment length polymorphism (TRFLP) of LAB communities; and 2) to explore the bacterial communities of wine fermentation to better understand the biodiversity of wine at a depth previously unattainable using other techniques. Analysis of amplicons from the V4 and V5 provided similar views of the bacterial communities of botrytized wine fermentations, revealing a broad diversity of low-abundance taxa not traditionally associated with wine, as well as atypical LAB communities initially detected by TRFLP. The V4 domain was determined as the more suitable read for wine ecology studies, as it provided greater taxonomic depth for profiling LAB communities. In addition, targeted enrichment was used to isolate two species of Alphaproteobacteria from a finished fermentation. Significant differences in diversity between inoculated and uninoculated samples suggest that Saccharomyces inoculation exerts selective pressure on bacterial diversity in these fermentations, most notably suppressing abundance of acetic acid bacteria. These results determine the bacterial diversity of botrytized wines to be far higher than previously realized, providing further insight into the fermentation dynamics of these wines, and demonstrate the utility of next-generation sequencing for wine ecology studies.

[1]  Nicholas A. Bokulich,et al.  Differentiation of mixed lactic acid bacteria communities in beverage fermentations using targeted terminal restriction fragment length polymorphism. , 2012, Food microbiology.

[2]  Nicholas A. Bokulich,et al.  Profiling the Yeast Communities of Wine Fermentations Using Terminal Restriction Fragment Length Polymorphism Analysis , 2012, American Journal of Enology and Viticulture.

[3]  Rob Knight,et al.  Selection of primers for optimal taxonomic classification of environmental 16S rRNA gene sequences , 2012, The ISME Journal.

[4]  Rob Knight,et al.  Comparison of Illumina paired-end and single-direction sequencing for microbial 16S rRNA gene amplicon surveys , 2011, The ISME Journal.

[5]  J. Leveau,et al.  Grapevine Microbiomics: Bacterial Diversity on Grape Leaves and Berries Revealed by High-Throughput Sequence Analysis of 16S rRNA Amplicons , 2011 .

[6]  G. Nychas,et al.  Bacterial species associated with sound and Botrytis-infected grapes from a Greek vineyard. , 2011, International journal of food microbiology.

[7]  Jaehyoung Kim,et al.  Resistant Starches Types 2 and 4 Have Differential Effects on the Composition of the Fecal Microbiota in Human Subjects , 2010, PloS one.

[8]  Robert C. Edgar,et al.  Search and clustering orders of magnitude faster than BLAST , 2010, Bioinform..

[9]  William A. Walters,et al.  Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample , 2010, Proceedings of the National Academy of Sciences.

[10]  William A. Walters,et al.  QIIME allows analysis of high-throughput community sequencing data , 2010, Nature Methods.

[11]  I. Andorrà,et al.  Effect of fermentation temperature on microbial population evolution using culture-independent and dependent techniques , 2010 .

[12]  V. Kunin,et al.  Wrinkles in the rare biosphere: pyrosequencing errors can lead to artificial inflation of diversity estimates. , 2009, Environmental microbiology.

[13]  C. Quince,et al.  Accurate determination of microbial diversity from 454 pyrosequencing data , 2009, Nature Methods.

[14]  James R. Cole,et al.  The Ribosomal Database Project: improved alignments and new tools for rRNA analysis , 2008, Nucleic Acids Res..

[15]  I. Andorrà,et al.  Effect of oenological practices on microbial populations using culture-independent techniques. , 2008, Food microbiology.

[16]  R. Knight,et al.  Error-correcting barcoded primers for pyrosequencing hundreds of samples in multiplex , 2008, Nature Methods.

[17]  F. Bushman,et al.  Short pyrosequencing reads suffice for accurate microbial community analysis , 2007, Nucleic acids research.

[18]  G. Nychas,et al.  Yeast Community Structures and Dynamics in Healthy and Botrytis-Affected Grape Must Fermentations , 2007, Applied and Environmental Microbiology.

[19]  J. Tiedje,et al.  Naïve Bayesian Classifier for Rapid Assignment of rRNA Sequences into the New Bacterial Taxonomy , 2007, Applied and Environmental Microbiology.

[20]  J. Foster,et al.  MiCA: A Web-Based Tool for the Analysis of Microbial Communities Based on Terminal-Restriction Fragment Length Polymorphisms of 16S and 18S rRNA Genes , 2007, Microbial Ecology.

[21]  G. Nychas,et al.  Yeast Populations Residing on Healthy or Botrytis-Infected Grapes from a Vineyard in Attica, Greece , 2007, Applied and Environmental Microbiology.

[22]  O. Claisse,et al.  Inventory and monitoring of wine microbial consortia , 2007, Applied Microbiology and Biotechnology.

[23]  James R. Cole,et al.  The ribosomal database project (RDP-II): introducing myRDP space and quality controlled public data , 2006, Nucleic Acids Res..

[24]  G. de Revel,et al.  Interactions between Brettanomyces bruxellensis and other yeast species during the initial stages of winemaking , 2006, Journal of applied microbiology.

[25]  Christopher J. Williams,et al.  Statistical methods for characterizing diversity of microbial communities by analysis of terminal restriction fragment length polymorphisms of 16S rRNA genes. , 2006, Environmental microbiology.

[26]  R. Knight,et al.  UniFrac: a New Phylogenetic Method for Comparing Microbial Communities , 2005, Applied and Environmental Microbiology.

[27]  D. Cowan,et al.  Review and re-analysis of domain-specific 16S primers. , 2003, Journal of microbiological methods.

[28]  M. Sipiczki Candida zemplinina sp. nov., an osmotolerant and psychrotolerant yeast that ferments sweet botrytized wines. , 2003, International journal of systematic and evolutionary microbiology.

[29]  G. Fleet Yeast interactions and wine flavour. , 2003, International journal of food microbiology.

[30]  L. Cocolin,et al.  Yeast Diversity and Persistence in Botrytis-Affected Wine Fermentations , 2002, Applied and Environmental Microbiology.

[31]  Andrew P. Martin Phylogenetic Approaches for Describing and Comparing the Diversity of Microbial Communities , 2002, Applied and Environmental Microbiology.

[32]  W. Wade,et al.  Design and Evaluation of Useful Bacterium-Specific PCR Primers That Amplify Genes Coding for Bacterial 16S rRNA , 1998, Applied and Environmental Microbiology.

[33]  Ronald M. Atlas,et al.  Handbook of microbiological media , 1993 .

[34]  P. Ribereau-gayon,et al.  Evolution of Acetic Acid Bacteria During Fermentation and Storage of Wine , 1984, Applied and environmental microbiology.

[35]  L. Cocolin,et al.  Direct Identification of the Indigenous Yeasts in Commercial Wine Fermentations , 2001, American Journal of Enology and Viticulture.

[36]  D. Faith Conservation evaluation and phylogenetic diversity , 1992 .

[37]  P. Ribereau-gayon New Developments In Wine Microbiology , 1985, American Journal of Enology and Viticulture.