Mapping microbial ecosystems and spoilage-gene flow in breweries highlights patterns of contamination and resistance

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.

Keywords: beer; built environment; droplet digital PCR; ecology; food fermentation; infectious disease; microbiology; next-generation sequencing; none.

Figure 1.. 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. DOI: http://dx.doi.org/10.7554/eLife.04634.003

Figure 2.. 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. DOI: http://dx.doi.org/10.7554/eLife.04634.004

Figure 3.. 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. DOI: http://dx.doi.org/10.7554/eLife.04634.005

Figure 4.. 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. DOI: http://dx.doi.org/10.7554/eLife.04634.006

Figure 5.. Spatial distribution heatmaps of fungi in brewery environments across seasons.

Plots indicate relative abundance of fungal taxa detected by ITS sequence reads across brewery surfaces at different times: Autumn (left), Spring (center), and Summer (right). See Figure 1 for a floorplan key and description of surfaces. Note that the floorplans change between seasons as some samples were only collected as specific timepoints and the wild brewing facility was built and opened during the Spring sampling time. DOI: http://dx.doi.org/10.7554/eLife.04634.007

Figure 6.. Spatial distribution heatmaps of bacteria in brewery environments across seasons.

Plots indicate relative abundance of bacterial taxa detected by 16S rRNA gene sequence reads across brewery surfaces at different times: Autumn (left), Spring (center), and Summer (right). Scales on right represent relative abundance scale (maximum 1.0) for each row of plots. See Figure 1 for a floorplan key and description of surfaces. Note that the floorplans change between seasons as some samples were only collected as specific timepoints and the wild brewing facility was built and opened during the Spring sampling time. DOI: http://dx.doi.org/10.7554/eLife.04634.008

Figure 7.. Spatial distribution heatmaps of bacteria in brewery environments across seasons (part 2).

Plots indicate relative abundance of bacterial taxa detected by 16S rRNA gene sequence reads across brewery surfaces at different times: Autumn (left), Spring (center), and Summer (right). Scales on right represent relative abundance scale (maximum 1.0) for each row of plots. See Figure 1 for a floorplan key and description of surfaces. Note that the floorplans change between seasons as some samples were only collected as specific timepoints and the wild brewing facility was built and opened during the Spring sampling time. DOI: http://dx.doi.org/10.7554/eLife.04634.009

Figure 8.. Lactic acid bacterial community composition on brewery surfaces, beers, and ingredients.

LAB-TRFLP profiles of samples exhibiting high Lactobacillales relative abundance by 16S rRNA gene sequencing. DOI: http://dx.doi.org/10.7554/eLife.04634.010

Figure 9.. Hop-resistance gene frequency on brewery surfaces and beers.

(A) ddPCR detection of hitA, horA, horB, and horC on surfaces (detected as copies/cm²) and in beers (copies/ml). Bar height indicates cumulative log gene abundance; colors indicate relative gene frequencies superimposed on these bars. Two barrel bung (stopper) samples are depicted on the left, one has no detection. (B) Pearson product-moment correlation matrix between hop-resistance genes, Lactobacillales relative abundance by 16S rRNA gene sequencing, and relative abundance of the dominant lactic acid bacteria detected by LAB-TRFLP. The color and shape of correlation ellipses (lower-left) indicate Pearson's product-moment correlation coefficient (r) between intersecting variables, as depicted in the key to the right. Correlations with larger positive r values are depicted as darker blue with increasingly narrow, upward-pointing ellipses. Correlations with larger negative r values are depicted as darker red with increasingly narrow, downward-pointing ellipses. Weaker correlations are depicted as wider, lighter colored ellipses. The corresponding p values for all correlation tests are provided in the reflected intersection (top-right). DOI: http://dx.doi.org/10.7554/eLife.04634.011