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Temporal and Spatial Distribution of the Acetic Acid Bacterium Communities throughout the Wooden Casks Used for the Fermentation and Maturation of Lambic Beer Underlines Their Functional Role.

ABSTRACT: Few data have been published on the occurrence and functional role of acetic acid bacteria (AAB) in lambic beer production processes, mainly due to their difficult recovery and possibly unknown role. Therefore, a novel aseptic sampling method, spanning both the spatial and temporal distributions of the AAB and their substrates and metabolites, was combined with a highly selective medium and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) as a high-throughput dereplication method followed by comparative gene sequencing for their isolation and identification, respectively. The AAB (Acetobacter species more than Gluconobacter species) proliferated during two phases of the lambic beer production process, represented by Acetobacter orientalis during a few days in the beginning of the fermentation and Acetobacter pasteurianus from 7 weeks until 24 months of maturation. Competitive exclusion tests combined with comparative genomic analysis of all genomes of strains of both species available disclosed possible reasons for this successive dominance. The spatial analysis revealed that significantly higher concentrations of acetic acid (from ethanol) and acetoin (from lactic acid) were produced at the tops of the casks, due to higher AAB counts and a higher metabolic activity of the AAB species at the air/liquid interface during the first 6 months of lambic beer production. In contrast, no differences in AAB species diversity occurred throughout the casks.IMPORTANCE Lambic beer is an acidic beer that is the result of a spontaneous fermentation and maturation process. Acidic beers are currently attracting attention worldwide. Part of the acidity of these beers is caused by acetic acid bacteria (AAB). However, due to their difficult recovery, they were never investigated extensively regarding their occurrence, species diversity, and functional role in lambic beer production. In the present study, a framework was developed for their isolation and identification using a novel aseptic sampling method in combination with matrix-assisted laser desorption ionization-time of flight mass spectrometry as a high-throughput dereplication technique followed by accurate molecular identification. The sampling method applied enabled us to take spatial differences into account regarding both enumerations and metabolite production. In this way, it was shown that more AAB were present and more acetic acid was produced at the air/liquid interface during a major part of the lambic beer production process. Also, two different AAB species were encountered, namely, Acetobacter orientalis at the beginning and Acetobacter pasteurianus in a later stage of the production process. This developed framework could also be applied for other fermentation processes.

SUBMITTER: De Roos J

PROVIDER: S-EPMC5861831 | biostudies-literature | 2018 Apr

REPOSITORIES: biostudies-literature

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Project description:Traditional lambic beer production takes place through wort inoculation with environmental air and fermentation and maturation in wooden barrels. These wooden casks or foeders are possible additional inoculation sources of microorganisms for lambic worts. To date, however, these lambic barrels have been examined only with culture-dependent techniques, thereby missing a portion of the microorganisms present. Moreover, the effects of the cleaning procedures (involving high-pressure water and/or fumigation) and the barrel type on the microbial community structures of the interior surfaces of wooden lambic barrels were unclear. The culture-dependent plating and culture-independent amplicon sequencing of swab samples obtained from the interior surfaces of different wooden casks and foeders used for traditional lambic beer production in Belgium revealed that the microbial compositions of these surfaces differed statistically throughout the barrel-cleaning procedures applied. At the end of the cleaning procedures, amplicon sequencing still detected fermentation- and maturation-related microorganisms, although only a few colonies were still detectable using culture-dependent methods. It is possible that some of the surviving microorganisms were missed due to the presence of many of these cells in a viable but not culturable state and/or engrained deeper in the wood. These surviving microorganisms could act as an additional inoculation source, besides brewery air and brewery equipment, thereby helping to establish a stable microbial community in the wort to diminish batch-to-batch variations in fermentation profiles. Furthermore, the microbial compositions of the interior barrel surfaces differed statistically based on the barrel type, possibly reflecting different characteristics of the lambic barrels in terms of age, wood thickness, and wood porosity.IMPORTANCE Although the coolship step is generally regarded as the main contributor to the spontaneous inoculation by environmental air of fresh worts for lambic beer production, it is known that microorganisms often associate with specific surfaces present in a brewery. However, knowledge about the association of microorganisms with the interior surfaces of wooden lambic barrels is limited. To clarify the role of casks and foeders as additional microbial inoculation sources, it was important to determine the influence of the barrel characteristics and the cleaning procedures on the microbial communities of the interior barrel surfaces. Moreover, this helped to elucidate the complex spontaneous lambic beer fermentation and maturation process. It will allow further optimization of the lambic beer production process, as well as the wooden-barrel-cleaning procedures applied.

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Temporal and Spatial Distribution of the Acetic Acid Bacterium Communities throughout the Wooden Casks Used for the Fermentation and Maturation of Lambic Beer Underlines Their Functional Role.

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