Acta agriculturae Slovenica, 116/2, 237–243, Ljubljana 2020 doi:10.14720/aas.2020.116.2.1692 Original research article / izvirni znanstveni članek The magic world of whiskey microbiota Ajda PRISTAVEC 1, 2 , Simon KOREN 3 , Barbara JERŠEK 4 , Anja VERONOVSKI 3 , Leon KOROŠEC 3 , Miha KOVAČ 3 , Minka KOV AČ 3 , Nataša TOPLAK 3 Received May 26, 2020; accepted October 03, 2020. Delo je prispelo 26. maj 2020, sprejeto 03. oktober 2020. 1 Université catholique de Louvain, Place de l’Université , Ottignies-Louvain-la-Neuve, Belgium 2 Corresponding author, e-mail: ajda.pristavec@student.uclouvain.be 3 Omega d.o.o., Ljubljana, Slovenia 4 University of Ljubljana, Biotechnical Faculty, Department of Food Science and Technology, Slovenia The magic world of whiskey microbiota Abstract: Modern metagenomics techniques in combi- nation with next generation sequencing are increasingly used for research of numerous environments inhabited by diverse microbiota. In the present study we focused on a rather unu- sual environment for their growth, a forgotten bottle of blended Scotch whiskey. Whiskey is a world-known popular spirit, tra- ditionally produced in a series of steps comprising malting of barley, fermenting the malt to an alcoholic wort, distilling and at least 3-year long maturation in oak casks, followed by filtra- tion. In the process, notably in the fermentation, microorgan- isms play a crucial role. However, we were primarily interested in potential microbiological and chemical changes that might have taken place over the years while the half-empty whiskey bottle was left open. We found that only a very low number of aerobic mesophilic bacteria survived in it while the ethanol content decreased from 40 % to approximately 30 %. Interest- ingly, the metagenomics analysis showed there was a large and diverse microbial community present in the forgotten whiskey. Among the most abundant microorganisms were members of human commensal microbiota, some potentially disease-caus- ing and also food spoiling bacteria, in particular genus Pseu- domonas. Surprisingly, we even found a non-negligible number of typically environmental bacterial species. Key words: whiskey; metagenomics; bacteria Čarobni svet mikrobiote viskija Izvleček: Raziskave raznovrstnosti mikrobiote številnih okolij so vse pogosteje izvedene s kombiniranjem naslednje ge- neracije sekvenciranja in metagenomskih metod. V naši študiji smo se osredotočili na nenavadno okolje za rast mikroorganiz- mov, in sicer pozabljeno steklenico Scotch viskija. Viski je sve- tovno poznana in priljubljena žgana pijača, ki je tradicionalno proizvedena v več korakih od pridobivanja ječmenovega slada, fermentacije v alkoholno sladico in destilacije, ki ji sledi vsaj 3 leta trajajoče staranje v hrastovih sodih in končna filtracija. Za fermentacijo so mikroorganizmi nujno potrebni. V naši raziskavi so nas predvsem zanimale potencialne mikrobiolo- ške in kemijske spremembe, ki so se zgodite tekom let, ko je bila pozabljena, pol prazna steklenica odprta. Ugotovili smo, da je preživelo le zelo malo števila aerobnih mezofilnih bakterij. Koncentracija etanola se je zmanjšala iz začetnih 40 % na pri- bližno 30 %. Metagenomska analiza je razkrila veliko in razno- vrstno mikrobno skupnost, ki je živela v pozabljenem viskiju. Med najpogostejšimi mikroorganizmi so bili člani običajne člo- veške mikrobiote, nekaj potencialno patogenih bakterij kot tudi kvarljivcev hrane, na primer bakterije rodu Pseudomonas. Pre- senetljivo smo potrdili prisotnost tudi nezanemarljivega število tipično okoljskih vrst. Ključne besede: viski; metagenomika; bakterije Acta agriculturae Slovenica, 116/2 – 2020 238 A. PRISTAVEC et al. 1 INTRODUCTION Microorganisms are extraordinarily diverse and well-conserved living beings that can be found in virtu- ally any habitat on the Earth, regardless of how hostile it may be. Recent advances of biomolecular techniques such as next generation sequencing (NGS) have made it possible to gain an important insight in microbial ge- nomes or their specific regions, especially coupled with metagenomics studies. Together with Fourier-transform infrared spectroscopy (FTIR), we can use them to closely inspect the diversity of bacteria and the composition of the medium they live in. Metagenomics is an analysis of genetic information from a microbial sample of a specific environment rather than from identical cells cultured in a dish (Laudadio et al., 2019). These sorts of studies are therefore particu- larly advantageous for the characterisation of groups of microorganisms that cannot be isolated, yet there might be thousands of different species of bacteria in a sample, of which many potentially closely related. There are dif- ferent NGS techniques on which metagenomics can rely, two primaries being amplicon based and shotgun (T essler et al., 2017). In the former, a specific region of genome such as 16S rRNA gene sequence is targeted, whereas in the later regions of randomly digested DNA are se- quenced (Laudadio et al., 2019). Each of the techniques has its own advantages and disadvantages. Recently, Tes- sler et al. (2017) have compared the two strategies in a large-scale study of microbial diversity and have shown that the 16S rRNA amplicon approach was superior in both phyla and family identification despite the fact that shotgun strategy produces over a hundred times more of reads. Furthermore, standard analytical FTIR spectros- copy method combined with Attenuated total reflection (ATR), or ATR-FTIR technique, can be used to study dif- ferent organic materials from liquid to solid samples and can give us qualitative as well as quantitative data about matrices such as whiskey. The central aim of the present study was to take ad- vantage of such metagenomics studies to characterise a potential microbial community in a forgotten bottle of blended Scotch whiskey. Whiskey is a popular mature spirit drink with a long history dating back to the 15 th century, originating from Scotland. It is traditionally pro- duced in a lengthy process which consists of malting of barley, fermenting the malt or some other grain to an al- coholic wort and of distilling, followed by at least 3-year long maturation in oak casks and filtration. Before bot- tling, whiskey is diluted by water to the desired alcohol concentration and to enhance its rich aroma (Karlsson & Friedman, 2017). For the fermentation step in the whiskey production, lactic acid bacterial community is of a great importance. The fact that the wort is not boiled makes it possible for the bacteria to survive and participate in the mixed yeast-bacteria fermentation (Makanjuola et al., 1992). At first, bacterial growth is heavily suppressed by the growth of the yeast, then the number of lactic acid bacteria (LAB) rises exponentially in an intermediate phase characterised by heterofermentative Lactobacil- lus such as Lb. fermentum Beijerinck, 1901 (van Beek & Priest, 2003), and is finally followed by a stationary phase where lactic acid continues to accumulate due to activ- ity of mostly homofermentative bacteria such as Lb. casei (Orla-Jensen, 1916) Hansen & Lessel, 1971 or Lb. para- casei Collins et al., 1989 (van Beek & Priest, 2002). The heterofermentative and homofermentative Lactobacillus flora was reported to comprise strains of Lb. fermentum, Lb. paracasei, Lb. brevis (Orla-Jensen, 1919) Bergey et al., 1934 and other more rarely detected Lactobacillus (Simp- son et al., 2001). However, microorganisms can be also responsible for spoilage of an alcoholic beverage. For ex- ample, in brewing, Gram-negative bacteria such as acetic acid bacteria, Zymomonas and Enterobacteriaceae are some of the common beer spoilers that require monitor- ing in the process (Paradh, 2015). Furthermore, a recent study has shown that Staphylococcus xylosus Schleifer & Kloos, 1975 makes the beer turbid and produces organic acids and biogenic amines, which alter the taste of the drink (Yu et al., 2019). Nonetheless, the spoilers of whis- key have not yet been described. Our studied sample was an abandoned half-empty 70 cl bottle of blended Scotch whiskey with 40.0 % etha- nol content, which was matured for at least 12 years be- fore put on sale. This Scotch whiskey was left open for a few years at the room temperature and a noticeable cloudy sediment had formed in it over time. From the microbiological standpoint whiskey appears to be far from a favourable medium for the growth of microor- ganisms, which intrigued us to take a closer look into biochemical alterations of the whiskey, in particular the microbial communities that might have sprouted in the bottle over the years. For this end, various approaches from standard analytical techniques such as ATR - FTIR spectroscopy, to microbiology and modern genetics were combined. 2 MATERIAL AND METHODS 2.1 MATERIAL In our study the original sample was a half-emp- ty70 cl bottle of blended Scotch whiskey. The bottle was originally marked as 12 years matured whiskey with 40 % of ethanol contents. The bottle was left open for a few Acta agriculturae Slovenica, 116/2 – 2020 239 The magic world of whiskey microbiota years at the room temperature. Over the time a notice- able cloudy sediment had formed at the bottom. In our study we used this turbid part of the whiskey. 2.2 METHODS 2.2.1 ATR – FTIR spectroscopy ATR - FTIR instrument Spectrum 100 (Perki- nElmer) was used for determination of ethanol con- centration in the whiskey sample in the spectral region 4000-450 cm -1 , using 4 cm -1 resolution and 4 scans. First mixtures of water and absolute ethanol (purity ≥ 99.8 %, Sigma - Aldrich) with known ethanol concentrations were prepared and then IR spectra were collected. By increasing ethanol concentration in reference samples (10 %, 20 %, 30 %, 40 %, 50 %, 100 %) broad peak in the range of 3000-3500 cm -1 decreases and peaks between 2850 and 3000 cm -1 , at 1045 cm -1 and at 1087 cm -1 in- crease. Ethanol concentration in whiskey could be deter- mined by comparison with other FTIR spectra or calcu- lated using Spectrum Quant program (PerkinElmer). 2.2.2 Culturing of bacteria Sample of the turbid part of the whiskey was anal- ysed for aerobic mesophilic bacteria and LABwith plate count method using appropriate medium and incuba- tion conditions. Plate Count Agar (PCA, Oxoid CM0325, Hampshire, England) was used for aerobic mesophilic bacteria and De Man, Rugosa, Sharpe medium (MRSc, Oxoid CM0361) with cycloheximide (Sigma Aldrich, 66- 81-9, St. Louis, MO, USA; 100 mg l -1 ) for LAB. Samples in aliquots of 1 ml were poured and mixed with media and agar plates were incubated for up to 10 days at 30  o C in normal atmosphere for aerobic mesophilic bacteria and in in anaerobic atmosphere in jar obtaining with anaero- bic atmosphere generation bag (Sigma Aldrich, 68061) for LAB. After incubation the number of colonies was counted, and results were expressed as average number of colony forming unit (CFU) per ml of sample (CFU/ ml). 2.2.3 Extraction of DNA and quantification DNA was extracted using PrepMan Ultra Sample Preparation Reagent following the manufacturer’s pro- tocol. 1 ml of the turbid part of the whiskey was first centrifuged for 3 min (3,000 g) and the supernatant was removed from the pellet. After DNA extraction the con- centration was measured with Qubit v4 and Qubit ds- DNA High-Sensitivity (HS) kit (all Thermo Fisher Sci- entific). 2.2.4 16S rRNA PCR amplification, Ion Torrent li- brary preparation and sequencing The seven hypervariable regions of 16S rRNA gene of bacteria were amplified in multiplex PCR reaction using the Ion 16S™ Metagenomics Kit. NGS library was prepared using Ion Plus Fragment Library Kit (Thermo Fisher Scientific), following the manufacturer instruc- tions. The amount and size distribution of the prepared library fragments were determined with the Labchip GX microfluidic capillary electrophoresis (PerkinElmer). Emulsion PCR and the enrichment steps were carried out using Ion PGM Hi-Q View OT2 Kit, as described by the manufacturer. Library templates were sequenced with on PGM Hi-Q View Sequencing Kit using an Ion 318 chip with the Ion T orrent PGM instrument (all Thermo Fisher Scientific). Signal processing and base calling were per- formed with the Torrent Suite software version 5.12. 2.2.5 Bioinformatics analysis Bioinformatics analysis was done with cloud-based software Ion Reporter 5.12 (Thermo Fisher Scientific). Only complete reads covering entire amplicons from primer to primer were used in the analysis. To avoid an artificial increase in sequencing diversity arising from random sequencing errors, only reads with 10 or more copies were used in the analysis. The reads which met all the criteria for the analysis were mapped to both the curated open-source Greengenes v13.5 database and the validated commercial MicroSEQ® 16S Reference Library v2013.1 (Thermo Fisher Scientific). Percentage identity cut-off values of 97 % and 99 % were used to assign gen- era and species, respectively. 3 RESULTS 3.1 FTIR ANALYSIS Ethanol concentration in whiskey was determined by comparison with other FTIR spectra or calculated us- ing Spectrum Quant software. By observing peaks from FTIR spectra, it can be concluded, that ethanol concen- tration in whiskey sample is around 30 % (Figure 1 and Figure 2). However, using Spectrum Quant software, Acta agriculturae Slovenica, 116/2 – 2020 240 A. PRISTAVEC et al. where Beer’s Law algorithm was used, calculated value of ethanol concentration in tested whiskey is 30.1 %. 3.2 CULTURING OF VIABLE BACTERIA The number of viable bacteria in whiskey was as expected very low, as the average number of aerobic me- sophilic bacteria was 45 ± 10 CFU ml -1 (Gram-positive cocci, and Gram-negative rods), and LAB were not found (< 10 CFU ml -1 ). 3.3 16S rRNA ANALYSIS In total, 212,571 reads were obtained after the default filtering and quality trimming in Torrent Suite Software. Of those, 186,267 reads covered the entire amplicon and were included in further analysis. After discarding reads with less than 10 copies, 92,554 were included in the mapping pipeline. 92,101 reads mapped to at least one of the databases, with only 453 reads remaining unmapped. Surprisingly, mapping revealed a diverse bacterial community in the sample. In total, 142 bacterial families, 149 genera and 159 species could be mapped unambigu- ously (Figure 3A and Figure 3B). More precise results show that Staphylococcus was the most abundant genus in the sample (21.6 % of mapped reads), followed by Anaerococcus (6.2 %), Methylobacte- rium (5.9 %) and Streptococcus (5.2 %). Family distribu- tion was more even, with Staphylococcaceae (14.1 %), Sphingomonadaceae (9.3 %), Enterobacteriaceae (9.0 %) and Comamonadaceae (6.9 %) representing the four most abundant operational taxonomic units (OTUs) at Figure 1: FTIR spectra of pure water, 100 % ethanol, mixtures of ethanol and water (10 %, 20 %, 30 % and 40 %) and whiskey sample in the range 1800-770 cm -1 . Figure 2: FTIR spectra of pure water, 100 % ethanol, mixtures of ethanol and water (10 %, 20 %, 30 % and 40 %) and whiskey sample in the range 3800-2600 cm -1 . Acta agriculturae Slovenica, 116/2 – 2020 241 The magic world of whiskey microbiota this level. At genus and family level, 42.9 % and 40.0 % of reads mapped to OTUs with relative abundance lower than 3 %, which again demonstrates the high diversity of the population. Bacteroides fragilis (Veillon & Zuber, 1898) Castellani & Chalmers, 1919, Campylobacter ureo- lyticus (Jackson & Goodman, 1978) Vandamme et al, 2010, Anaerococcus vaginalis (Li et al., 1992) Ezaki et al., 2001, Morganella morganii (Winslow et al., 1919) Fulton, 1943, Anaerococcus octavius Murdoch et al., 1997) Ezaki et al., 2001and Finegoldia magna (Prevot, 1933) Murdoch & Shah, 2000 were the most abundant species detected. This does not match data from higher taxonomic units but is expected for 16S metagenomic analysis. In contrast to common strategies that focus only on one or two re- gions (V3, V4), our approach covered sequencing of 7 variable 16S regions, to ensure as much discriminatory power as possible and economically feasible. Neverthe- less, for many OTUs, similarities in sequences of 16S rRNA genes for many species are still so high, that it is impossible to distinguish them at the level of species. The most abundant genus Staphylococcus represents such ex- ample in our analysis. 4 DISCUSSION The abandoned bottle of Scotch whiskey might seem as a rather hostile medium for the growth of microorgan- isms, yet our metagenomics analysis suggests there was a large community present in it. Even though the ethanol content of the whiskey decreased from 40 % to 30.1 %, diverse species of bacteria had proliferated in it before dying off. Using the cut-off of at least 10 reads, we identi- fied around 160 different species from roughly 150 gen- era in our whiskey sample. The highest number of reads was recorded for the family of Gram-positive bacteria Staphylococcocae, all of them from genus Staphylococcus. The most abundantly represented species was Staph. Epi- dermidis (Winslow & Winslow, 1908) Evans, 1916 which is universally present on human skin and mucosa but can also act as opportunistic pathogen (Coates et al., 2014). Taxonomic composition analysis also revealed very high number of reads for exceptionally large family of Gram- negative bacteria Enterobacteriaceae, which comprises some potentially disease-causing genera such as Shigella, Salmonella, Klebsiella and Escherichia (Brenner et al., 2005). Amongst the most commonly detected species in the whiskey sample were predominantly commensal or- ganisms which inhabit human alimentary tract or colo- nise our skin and mucous membranes, some being po- tentially pathogenic. For instance, the most abundantly represented species Bacteroides fragilis are obligate an- aerobic Gram-negative bacteria that are a normal part of Figure 3: Distribution of (A) families a (B) genera unambiguously detected in the whiskey sample at relative abundances of 3 % or higher Acta agriculturae Slovenica, 116/2 – 2020 242 A. PRISTAVEC et al. human intestinal microbiota, yet become pathogenic by endotoxin secretion when the mucosal barrier is disrupt- ed (Elsaghir & Reddivari, 2020). Similarly, a facultative- anaerobic member of Enterobactericeae family, Morgan- ella morganii is a common member of human intestinal tract flora, but can also act as opportunistic pathogen causing wound and urinary tract infections (Liu et al., 2016). Another frequently detected Gram-negative an- aerobic species is Campylobacter ureolyticus, which has been identified as gastrointestinal pathogen, causing Campylobacter-related gastroenteritis (O’Donovan et al., 2014). Furthermore, also aerobic Gram-positive bac- teria such as those from genus Corynebacterium were highly represented in the whiskey sample, in particu- lar Corynebacterium striatum (Chester, 1901) Eberson, 1918. This species commonly colonises skin and mucous membranes, but has also been identified as an emerging pathogen causing amongst other also bone and joints in- fections (Noussair et al., 2019). On the other hand, food spoilers were not as abun- dantly represented in the microbial community of our abandoned bottle. Remenant et al. (2015) described LAB and genera Clostridium, Serratia, Hafnia, and Pseu- domonas as the main known food spoilers. One impor- tant example highly present in our whiskey is genus Pseu- domonas, which largely contributes to the food spoilage process. Pseudomonas comprise thermotolerant spoilage microorganisms that cause off-flavour release, reduction of shelf-life and quality of dairy and meat products, dis- coloration of dairy products and browning of vegetables, along with biofilm formation on chilled foods (Quintieri et al., 2019). Regarding LAB, only very low number of reads were recorded for Lactococcus lactis (Lister, 1873) Schleifer et al., 1986 and even fewer for Lactobacillus in- ers Falsen et al., 1999, but we also recorded presence of Enterococcus faecalis (Andrewes & Horder, 1906) Schle- ifer & Kilpper-Balz, 1984 which is involved in greening and spoilage of meat (Remenant et al., 2015). Taking into account these metagenomic findings, it comes as no sur- prise that no viable LAB were detected in the anaerobic culture. Curiously, in the whiskey bottle there were also some species more characteristic for environmental sam- ples such as Rubrivivax gelatinosus (Molisch, 1907) Wil- lems et al., 1991, a purple nonsulfur photosynthetic bac- terium found for instance in freshwater ponds and food processing wastewater (Nagashima et al., 2012). Similar- ly, Diaphorobacter nitroreducens Khan & Hiraishi, 2003, a denitrifying microorganism, is also an example of bac- terium that has been previously isolated from activated sludge used in wastewater treatment (Khan & Hiairishi, 2002). Third such microorganism in our studied sample are aerobic, nitrite-oxidizing bacteria of genus Nitrospira which are abundant in various ecosystems such as waste- water treatment plants, freshwater, soils, groundwater and geothermal springs (Mehrani et al., 2020). 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