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60 P R A C T I C A L W I N E R Y & V I N E YA R D September 2015 T he use of sulfur dioxide (sulfites or SO 2 ) in winemaking is hardly a new technology— it has been used since the Romans started burning sulfur candles in emptied bar- rels to keep them from turning sour. The general antimicrobial and antioxidant effects of SO 2 in wine have also been well known for several decades, and many modern winemakers find SO 2 to be a key additive for producing (and preserving) premium quality wine. At crushing/pressing, the addition of SO 2 is thought to inhibit growth of cer- tain non-Saccharomyces yeasts and lactic acid bacteria (LAB), which can contribute to spoilage and problematic fermenta- tions. At adequate levels (approximately 50 ppm) SO 2 also reduces the activity of the oxidative enzyme polyphenol oxi- dase, which can rapidly deplete oxygen in juice/must at the expense of the grow- ing yeast population during the early stages of fermentation. What is not well established is precisely how SO 2 added early on shapes the micro- bial diversity of a wine fermentation. N.A. Bokulich et al. (2015) addressed this issue using some of the latest methods in microbial ecology. Traditional culture- based methods for examining microbial profiles of wines, while powerful and use- ful for many investigations, are limited in both their sensitivity and scope. These techniques are slow, laborious and not sensitive to the presence of very small or difficult-to-cultivate populations. For this investigation, high-through- put marker gene sequencing was used to examine the changes in both bacte- rial and fungal communities throughout the fermentation of a Chardonnay wine. The method begins with environmental samples — in this case fermenting must/ wine — whereby the total DNA was extracted and two separate sets of prim- ers (one bacterial and one fungal) were used in separate polymerase chain reac- tions (PCR) to amplify the target regions of all microbes present. For bacteria, this region was the V4 domain of the 16S rRNA genes, and for fungi the internal transcribed spacer (ITS) 1 loci were amplified. These two "ampli- cons" were then sequenced by modern techniques (in this case an Illumina MiSeq device), resulting in hundreds of thousands of short DNA sequences that were then quality filtered and classified into operational taxonomical units (OTUs) prior to identification and cataloging of the various microbial taxa by comparison to existing databases. What this yields, in the end, is the rela- tive frequencies of dozens of different types of microbes in each sample — a model of the microbial communities. Further statistical analyses allowed com- parison of samples to find the significant differences that exist among them. It is important to note that this process is based on DNA and does not differentiate between live or dead cells, nor those that might lie somewhere in between — in a viable but nonculturable (VBNC) state. However, its great strength lies in its sensitivity to even small, and oth- erwise difficult to detect, populations. Even more important, it is a rapid and affordable way to obtain a much more complete description of the microbial communities of hundreds (or thousands) of samples at once. How was the experiment conducted? The actual Chardonnay fermentations were conducted in triplicate 5-gallon fer- mentations, uninoculated at 0, 15, 20, 25, W I N E M A K I N G Michael L. Swadener 1 and David A. Mills 1,2 1 Department of Viticulture & Enology, 2 Department of Food Science & Technology, University of California, Davis BY How SO 2 Additions Influence Microbial Diversity During Fermentation NEW LOOK AT AN OLD PRACTICE How does SO 2 added early in the winemaking process impact the microbial diversity of a fermentation? T E C H N I C A L R E S O U R C E F O R G R O W E R S & W I N E R I E S Don Neel, Editor practicalwinerylibrary.com Access Practical Winery & Vineyard article archives online. SO 2 mainly impacted bacterial diversity, as fungal populations across the range of sulfite additions did not appear to differ significantly from one another throughout the course of the fermentations. Bacterial diversity, on the other hand, was found to be significantly impacted by different doses of SO 2 .