Author response: Mapping microbial ecosystems and spoilage-gene flow in breweries highlights patterns of contamination and resistance (2015)

Full text Figures and data Side by side Abstract eLife digest Introduction Results and discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Distinct microbial ecosystems have evolved to meet the challenges of indoor environments, shaping the microbial communities that interact most with modern human activities. Microbial transmission in food-processing facilities has an enormous impact on the qualities and healthfulness of foods, beneficially or detrimentally interacting with food products. To explore modes of microbial transmission and spoilage-gene frequency in a commercial food-production scenario, we profiled hop-resistance gene frequencies and bacterial and fungal communities in a brewery. We employed a Bayesian approach for predicting routes of contamination, revealing critical control points for microbial management. Physically mapping microbial populations over time illustrates patterns of dispersal and identifies potential contaminant reservoirs within this environment. Habitual exposure to beer is associated with increased abundance of spoilage genes, predicting greater contamination risk. Elucidating the genetic landscapes of indoor environments poses important practical implications for food-production systems and these concepts are translatable to other built environments. https://doi.org/10.7554/eLife.04634.001 eLife digest Many microbes—including bacteria and fungi—can affect the food and drink we consume, for better and for worse. Some spoil food, making it less tasty or even harmful to health. However, microbes can also be important ingredients: for example, yeast ferments malted barley sugars to make the alcohol and flavor of beer. Nowadays, many beers are made under carefully controlled conditions, where the only microbes in the beer should be the strain of yeast added to the barley sugars. A more traditional 'coolship' method can be used to make sour beers; the barley sugars cool in an open-topped vessel and are fermented by the yeast and bacteria found naturally on the raw ingredients and in the surrounding environment. Relatively little was known about how microbes spread around and adapt to living inside buildings. Now, Bokulich et al. have used a range of molecular and statistical techniques to examine how bacteria and fungi are dispersed throughout a North American brewery that produces beer using both conventional and coolship brewing techniques. Most of the microbes found in the building originated from the raw ingredients used to make the beer, with different parts of the brewery containing different species. Over the course of a year, some species spread to new parts of the building; a statistical method predicted the sources of these microbes, and revealed some key areas and features of the brewery that affect microbial transfer. Bokulich et al. also looked at the spread of genes that enable their bacterial hosts to spoil beer, including those that protect bacteria from the antimicrobial action of the hops that flavor many beers. Lactic acid bacteria are the main cause of beer spoilage and so are usually to be avoided in breweries, but are also a normal ingredient in sour beer. In the brewery Bokulich et al. investigated, beer-spoilage and hop-resistance genes were found throughout the brewery, even in areas not used to produce sour beer. However, little beer spoilage occurred. The techniques used by Bokulich et al. to track the spread of microbes and their detrimental genes could be used in the future to understand how microbes adapt to other indoor environments. Indeed, Bokulich et al. suggest that breweries could be used as models to safely understand the factors that influence microbial movement in any food-production facility as well as other building environments. https://doi.org/10.7554/eLife.04634.002 Introduction Microbial activity is an inherent feature of food-processing systems, influencing the quality and healthfulness of foods for human consumption. Like other indoor environments, the building materials, substrates, and physiochemical conditions encountered by microbes in food-processing facilities differ dramatically from the outdoor conditions to which microbial life evolved (Kelley and Gilbert, 2013). How this has impacted the adaptation and ecological assemblages of microbes in food systems is largely unexplored. While the microbial communities of other indoor environments, such as homes (Lax et al., 2014), office buildings (Kembel et al., 2014), and hospitals (Bokulich et al., 2013a), can influence the health of their inhabitants, food-production facilities represent a unique type of built environment wherein microbial activities are intimately tied to product-quality outcomes. Thus, the sensory and safety effects of microbial growth in food-production streams have a much broader impact, such that consumer enjoyment and health can be linked to the hygiene and processing decisions at the food facility. Furthermore, both beneficial and detrimental microbial activities are well defined in foods, making these systems a useful model for exploring microbial ecosystem dynamics beyond the scope of contaminant mitigation. Fermented foods, including beer, have the additional distinction that microbial activity is central to their production, responsible for necessary transformations as well as product spoilage (Bokulich et al., 2012a; Bokulich and Bamforth, 2013). Most modern food-fermentation practices occur under relatively aseptic conditions, employing pure starter cultures in their production, and environmental contamination represents a prevalent threat to product integrity (Bokulich and Bamforth, 2013). Beer is typically produced through fermentation of malted barley sugars (wort) to alcohol by pure strains of Saccharomyces cerevisiae or Saccharomyces pastorianus, and any additional organisms—including cross-contamination from different Saccharomyces strains used in the same facility—are considered contaminants (Bokulich and Bamforth, 2013). In traditional fermentation production practices, conversely, microbial communities introduced from raw materials and processing environments are central to the fermentation process. For example, in the production of coolship (lambic-style) beers, a type of sour beer, no starter cultures are added to the wort; instead, boiled wort is allowed to cool overnight in a shallow, open-top vessel known as a coolship where indigenous microbiota are presumably introduced to the product, initiating fermentation (Van Oevelen et al., 1977; Bokulich et al., 2012b; Spitaels et al., 2014). The unique succession of indigenous microbiota in coolship ale fermentations sets them apart chemically and sensorially from other beers (Van Oevelen et al., 1976; Spaepen et al., 1978), making coolship breweries a particularly interesting system for tracking microbial populations in food-processing environments. Microbial spoilage genes are also well defined in brewing environments and can be studied in situ without representing a direct threat to public health. Beer is protected from wholesale microbial contamination through its alcoholic, low-pH, and antimicrobial properties, and modern sanitary technologies and practices minimize the threat of spoilage organisms that have evolved specifically to grow in beer (Bokulich and Bamforth, 2013). Nevertheless, biofilms and other environmental reservoirs remain potential sources of microbial contamination in breweries (Timke et al., 2005; Storgards et al., 2006; Timke et al., 2008; Mamvura et al., 2011; Matoulkova et al., 2012). As wort is boiled prior to fermentation, the primary reservoir for spoilage microorganisms in beer production is the brewery environment. Lactic acid bacteria (LAB) are of particular concern, as some members of this clade resist hop antimicrobial compounds, enabling growth and spoilage of beer through acidification, hazes, and off-flavors (Suzuki et al., 2006; Bokulich and Bamforth, 2013). Hop iso-alpha-acids, the primary antimicrobial compounds in beer, kill most Gram-positive bacteria by acting as ionophores, dissipating proton-motive force across cell walls (Simpson and Fernandez, 1992, 1994; Simpson, 1993a), and via oxidative stress (Behr and Vogel, 2010). Very few resistance mechanisms have been proposed (Suzuki et al., 2006), primarily being the multi-drug transporters horA (Sami et al., 1997; Sakamoto et al., 2001) and horC (Suzuki et al., 2005; Iijima et al., 2006), the transcriptional regulator of horC, horB (Suzuki et al., 2005; Iijima et al., 2006), and hitA, a divalent cation transporter (Hayashi et al., 2001) that imports manganese into the cell. These genes are all located on plasmids and transmit via horizontal gene transfer (Suzuki et al., 2005, 2006). The frequency and transmission of these and other beer-spoilage genes within processing environments—or any other reservoir for spoilage microbes—have yet to be tested. Here, we employ mixed molecular approaches (Bokulich and Mills, 2012b) and a Bayesian modeling method to interrogate the seasonal sources, reservoirs, and transmission of bacteria, fungi, and beer-spoilage genes within a North American brewery over the course of one year. Given the inherent role of environmental microbiota in conducting coolship ale fermentations, this serves as a model system for studying mechanisms for microbial transfer within food-processing systems. Information on population and gene flow extends to other food-processing systems where environmental microbiota are involved positively or negatively in the production, stability, and safety of human nutrition. Results and discussion The microbial ecosystems of breweries are inherently linked to successful product outcomes, impacting the qualities and healthfulness of beer. In conventional brewing, any microbe not intentionally inoculated is a contaminant (Bokulich and Bamforth, 2013). Conversely, in coolship ale and other sour beer brewing, adventitious microbiota are integral to the process. Thus, we studied microbial ecosystem dynamics in a brewery that produces conventional, sour, and coolship beers, in order to observe food–ecosystem interactions from a dual perspective. First, how do microbial community assemblages and spoilage-gene frequencies change over time with respect to contamination issues in a conventional brewery? Second, how do these same elements interact with the production of sour beers? Production environment microbiota are driven by substrate contact Short-amplicon marker-gene sequencing was employed to survey the bacterial and fungal consortia inhabiting the entire brewery environment. A total of 501 samples were collected during three seasons, representing the main processing surfaces and equipment used throughout the brewing process (Figure 1). Beta-diversity (between sample) comparisons provide useful assessments of the taxonomic similarity between different sites. Bray–Curtis dissimilarity of complete microbial profiles reveals that many samples cluster by processing room and substrate type regardless of season (Figures 2–3). Thus, fermenter samples cluster, associated with Bacillaceae; cellar production areas, associated with Micrococcaceae (including the beer-spoiling genera Kocuria and Micrococcus); wort, malt, and hotside (wort-preparation) surfaces, associated with Enterobacteriaceae, Leuconostocaceae, Candida santamariae, Pichia, and Rhodotorula; barrel-room floor samples, associated with Staphylococcaceae and Carnobacteriaceae; and beer samples, associated with Lactobacillaceae and Enterobacteriaceae. Barrels cluster, associated with Aspergillus, Eurotium, and Penicillium; coolship and barrel-room samples with Cryptococcus and Cladosporium. These taxonomic trends each demonstrate significant site associations (Kruskal–Wallis Bonferroni-corrected p < 0.05). S. cerevisiae was common throughout the brewery, but especially in the fermentation cellar. LAB and acetic acid bacteria were found sporadically at different sites and times, including on and near packaging equipment and fermenters inoculated with LAB (Figure 3). Figure 1 Download asset Open asset Brewery map and simplified brewing process diagrams. (A) Floorplan of brewery details surface sampling key and indicates separate sections of the brewery. LAC/Br fermenters indicate they were inoculated intentionally with lactic acid bacteria or Dekkera spp., respectively, at the time of sampling. (B) Process diagram for conventional beer brewing, illustrating the relationship between brewing stages and sections of the brewery. Grain is milled and taken to the brewhouse (hotside) area where it is mashed (steeped in hot water) to form wort, which is lautered (extracted from the grain by filtering and spraying with hot water) and then boiled with hops. Boiled wort is cooled and pumped to the fermentation cellar where it is inoculated with Saccharomyces and fermented. Optionally, barrel-aged beers are transferred to barrels after fermentation. Finished beers are transferred to conditioning tanks in a separate section of the brewery where they are cooled, carbonated, and then packaged. (C) Process diagram for coolship beer brewing. Same as conventional, but following boiling wort is pumped to the coolship room where it is left to cool overnight, exposed to the atmosphere. The following morning, the wort is pumped to barrels in which it is fermented and aged for 1–3 years. In the Autumn samples, this occurred in the barrel room in the main brewery, but in Spring and Summer this moved to a newly built facility dedicated to sour beers. All coolship and sour beers were packaged on separate equipment in this second facility. The distinction between coolship beers and sour beers is the use of this coolship; other sour beers are produced using conventional brewing methods (panel B), but are fermented with organisms other than Saccharomyces yeasts. https://doi.org/10.7554/eLife.04634.003 Figure 2 Download asset Open asset Taxon abundance heatmaps depict genus-level relative abundance of fungi across sampling sites detected by marker-gene sequencing. The relative abundances (RA) of each genus (columns) within each sample (rows) are indicated by the color of the intersecting tile. Sample types are indicated by colored bars to the left of each row, classified according to the location within the brewery (Figure 1) or the type of substrate (grain, wort, hops, beer). Dendrograms represent Bray–Curtis dissimilarity between samples (vertical trees) and shared-niche similarity between taxa (horizontal trees), respectively indicating taxonomic composition similarities and taxon co-occurrence patterns. Only taxa ≥0.05 relative abundance in at least one sample are shown. https://doi.org/10.7554/eLife.04634.004 Figure 3 Download asset Open asset Taxon abundance heatmaps depict family-level relative abundance of bacteria across sampling sites detected by marker-gene sequencing. The relative abundances of each genus (columns) within each sample (rows) are indicated by the color of the intersecting tile. Sample types are indicated by colored bars to the left of each row, classified according to the location within the brewery (Figure 1) or the type of substrate (grain, wort, hops, beer). Dendrograms represent Bray–Curtis dissimilarity between samples (vertical trees) and shared-niche similarity between taxa (horizontal trees), respectively indicating taxonomic composition similarities and taxon co-occurrence patterns. Only taxa ≥0.05 relative abundance in at least one sample are shown. https://doi.org/10.7554/eLife.04634.005 To further examine this relationship, the Bayesian technique sourcetracker (Knights et al., 2010) was used to test whether raw substrates may act as sources for the microbial consortia of brewery surfaces. This tool predicts the relative proportion of contamination in sink samples (in this case brewery surfaces) from microbial sources (raw ingredients and extraneous sources). Raw substrates (grain, hops, yeast, beer) and extraneous sources (human skin, outdoor air, soil, saliva, feces, freshwater, ocean water) from previous studies (Caporaso et al., 2011; Bowers et al., 2012; Caporaso et al., 2012; Bowers et al., 2013) were tested as microbial sources. Results reveal distinct patterns of contamination across seasons (Figure 4). Grains were predicted as the largest microbial contributor to hotside areas, almost all surfaces in the coolship room, and areas of the cellar away from the main fermentation area. Hops were predicted as the major contributor to cellar fermentation areas and fermentation equipment. Yeast was predicted as the highest contributor to fermenters, conditioning tanks, and packaging equipment. Beer was predicted to be a common contaminant around fermenters and barrels within the cellar. Skin was a minor contributor to some surfaces in the cellar. Other surfaces, including most barrels and barrel-room surfaces, were most influenced by unknown sources. Outdoor air, soil, saliva, feces, freshwater, and ocean water were predicted to provide only a very low level of contamination (<0.001 relative abundance). These results suggest that raw substrates are the main contaminant sources within the brewery, compared to extraneous sources. However, it is important to note that these predictions do not indicate a causative role for contamination. These predicted source/sink relationships could alternatively suggest that both are actually contaminated by another, untested source (e.g, fruit flies or other vectors could transfer microbes between these and other surfaces within the brewery). Nevertheless, these predictions highlight potential sources of contamination, or at least shared microbial transmission patterns, between substrates and equipment/surfaces within the brewery. Figure 4 Download asset Open asset Mapping microbial contamination sources inside the brewery. Floorplans of brewery indicate the predicted relative contamination of brewery surfaces by microbial sources (grains, hops, yeast, beer, human skin, unknown) at each season, estimated by SourceTracker (Knights et al., 2010). Coloration of each surface indicates the relative degree of microbial contamination from that source type (as indicated by keys to the right of each row; units are relative abundance). Contamination from outdoor air, soil, feces, freshwater, ocean water, and saliva was negligible (<0.001 relative abundance) and are not shown. See Figure 1 for a floorplan key and description of surfaces. https://doi.org/10.7554/eLife.04634.006 These findings suggest that substrate contact drives the microbial communities of different brewery surfaces. Consequently, raw materials (grain, hops, yeast) may be important vectors for any spoilage organisms encountered in the facility. This substrate-centric community structuring has also been observed in other food facilities (Bokulich and Mills, 2013a; Bokulich et al., 2013b, 2014). However, the brewery surface niches clustered throughout time with only subtle changes across seasons, unlike wineries, which were significantly impacted by seasonal factors (Bokulich et al., 2013b). This reflects the divergent production schemes of these two foods: whereas most beers are produced year-round, wine is an inherently seasonal product. The raw materials for beer are stored and processed throughout the year in a continual production schedule, creating a stable environment on equipment surfaces that interact with these same raw substrates on a daily basis. Instead, brewery environments may function more like cheese-making environments, where facility-specific 'house' microbial communities form on equipment surfaces in response to idiosyncrasies in the indoor environment (Bokulich and Mills, 2013a). Such a possibility would have interesting implications for sour beer breweries, and comparative studies of lambic and coolship breweries could offer insight into the brewery-specific flavor profiles displayed in these beers. Physical mapping illustrates microbial dispersal in processing facility By mapping surface samples to their physical location within the brewery, a spatial model emerges of microbial dispersion across brewery surfaces over time (Figures 5–7). Several populations visibly spread with time, most prominently S. cerevisiae, which progressed from dominating fermentation and packaging areas only in Autumn to being the most abundant fungus detected across the brewery in Spring and Summer. Likewise, C. santamariae displayed high abundance on hotside surfaces in Autumn but became more abundant and spread to nearby sections of the cellar in Spring and Summer (Figure 5). As building measurements were not taken during these times, the factors driving these changes cannot be assessed with these data, but warming temperatures and increasing humidity in the Spring and Summer months could be associated phenomena. Other taxa demonstrated more localized patterns of dispersion, such as Micrococcus and Kocuria, which appeared to spread the most around floors and other surfaces in cellar, barrel room, and packaging areas (Figure 6). Here, regular contact with beer runoff diluted with rinse water may support growth of these populations, which are associated with spoilage in low-alcohol beers (Back, 1981). High populations of Acetobacter and Lactobacillus were found more disparately, and specifically in areas where high volumes of wort and beer may be encountered: on conveyor belts and floors below packaging areas, hotside and cellar area sinks, and on sample ports for isolated fermenters and kegs (Figure 6). On these sites microbial communities can contact undiluted beer, for example, drips in the basin below the packaging-line belt, wort and beer collected for specific gravity (sugar concentration) measurements then in sinks, and sampling ports on This the known of these bacteria, which can spoil beers under conditions (Bokulich and Bamforth, 2013). Figure Download asset Open asset heatmaps of fungi in brewery environments across indicate relative abundance of fungal taxa detected by across brewery surfaces at different Autumn Spring and Summer See Figure 1 for a floorplan key and description of surfaces. that the change between seasons as some samples were only collected as specific and the brewing facility was built and during the Spring sampling Figure Download asset Open asset heatmaps of bacteria in brewery environments across indicate relative abundance of bacterial taxa detected by gene across brewery surfaces at different Autumn Spring and Summer on right represent relative abundance for each of See Figure 1 for a floorplan key and description of surfaces. that the change between seasons as some samples were only collected as specific and the brewing facility was built and during the Spring sampling Figure Download asset Open asset heatmaps of bacteria in brewery environments across seasons indicate relative abundance of bacterial taxa detected by gene across brewery surfaces at different Autumn Spring and Summer on right represent relative abundance for each of See Figure 1 for a floorplan key and description of surfaces. that the change between seasons as some samples were only collected as specific and the brewing facility was built and during the Spring sampling These results the of microbes in and time within a food-processing environment. not only with specific substrates, they patterns of dispersion within of the brewery. This useful insight into the transmission of these and especially taxa associated with beer through physical Microbial within that physical and physiochemical conditions humidity and can the spread of some surfaces that beer such as sinks, and are typically only by and may substrates for microbial growth and and occur in a production brewery floors is one increasing the for microbial spread and from these surfaces other surfaces, including equipment. and can also the of dispersal between these surfaces from one room to Physical to this as observed in this brewery where may the for contamination. This is particularly important in packaging areas, where microbial dispersal can sanitary surfaces and increasing the of spoilage in product. packaging from cellar and hotside and using such as may protect packaging surfaces and beer from such routes of contamination. into the relationship between building materials, spatial indoor conditions and and the and of microbial dispersion could important findings for brewery from the of microbial These findings are but should not be with they microbial dispersal in a brewery that from any of product contamination. Lactic acid bacteria profiles are also driven by substrate exposure species of LAB are the spoilage bacteria in beer fermentations as well as important members of sour beer However, gene are to (Bokulich and Mills, we used (Bokulich and Mills, to and LAB community in a of This raw and beer samples, as well as surfaces on which of by marker-gene sequencing was particularly Results indicate that different surface and sample types distinct lactic acid bacterial patterns, to the substrates encountered at that site or in that sample (Figure samples a of Lactobacillus Lactobacillus Lactobacillus and most of which were only detected in other and beer Many of these species are also found in beers (Bokulich and Bamforth, but associated with their in and sour beers (in this case coolship beers produced from different wort were by previous studies of coolship beers in this brewery (Bokulich et al., and barrel surfaces that these fermentations near the time of sampling Lactobacillus and Lactobacillus A were more common on these surfaces than in the beers or on other surfaces. Other sour and barrel-aged beers unique with of other Lactobacillus species only detected in coolship beers. and packaging area surfaces a more of but primarily the and detected in the wort and beer only was detected on grain samples, only could be from these samples, low LAB populations or of by grain the of less abundant Hop samples also a of different LAB populations, including and Figure Download asset Open asset Lactic acid bacterial community composition on brewery surfaces, beers, and profiles of samples high relative abundance by gene sequencing. These results that substrate drives the composition of LAB communities as well as microbial communities and highlight the of cross-contamination between different equipment surfaces. The of