Carbohydrate Metabolism Differentiates Pectinatus and Megasphaera Species Growing in Beer

Abstract

Obligate anaerobic beer spoilage bacteria have been a menace to the brewing industry for several decades. Technological advances in the brewing process aimed at suppressing aerobic spoilers gave rise to problems with obligate anaerobes. In previous studies, the metabolic spectrum of Pectinatus and Megasphaera species has been described, but their metabolism in the beer environment remains largely unknown. We used high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) and headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GCMS) to further characterize beer spoiled by 30 different strains from six beer-spoiling species of Pectinatus and Megasphaera (P. cerevisiiphilus, P. frisingensis, P. haikarae, M. cerevisiae, M. paucivorans, and M. sueciensis). We detected differences in carbohydrate utilization and the volatile organic compounds (volatilome) produced during beer spoilage by all six species. We were able to show that glycerol, one of the basic components of beer, is the common carbon source used by all strains. It appears that this carbon source allows for anaerobic beer spoilage by Pectinatus and Megasphaera despite the spoilage-preventing intrinsic barriers of beer (iso-α-acids, ethanol, low pH, scarce nutrients); thus, extrinsic countermeasures are key for prevention.

Keywords: Megasphaera spp.; Pectinatus spp.; beer spoilage; carbohydrate metabolism; volatile fatty acids.

Figure 1

Composition of sugars in the utilized commercial lager beer from triplicates. Error bars indicate the standard deviation for each compound.

Figure 2

Change in pH in triplicates of lager beer spoiled by the respective strains’ initial lager beer pH of 4.3. Strains are sorted by the difference in pH compared with the control beer. A superscript T indicates the respective type strain, and error bars represent the standard deviation.

Figure 3

Principal component analysis of the residual carbohydrates in all samples spoiled by the strains as well as in the control beer revealing three main clusters. On the top left, all Megasphaera strains cluster in close proximity to the control beer, fortifying the marginal utilization of all carbohydrates by strains of the genus. The bottom cluster harbors most strains of the genus Pectinatus that show a significant but similar utilization of glycerol and maltose, whereas the P. frisingensis strains in the upper right corner show the highest degradation of maltose and therefore group as a separate cluster. All samples were biological triplicates that were incubated for 23 days at 30 °C, and the control was an unspoiled lager beer treated the same way as the spoiled samples.

Figure 4

Principal component analysis revealing a genus specificity of the volatilomes of both Pectinatus spp. and Megasphaera spp. The difference in Pectinatus species is caused by the production of organic acids and diverse volatile compounds. All M. cerevisiae are distinctly clustered, and M. paucivorans and M. sueciensis are separated because of a more diverse production of sulfuric compounds. All samples were biological triplicates that were incubated for 23 days at 30 °C, and the control was an unspoiled lager beer treated the same way as the spoiled samples.