The Interior Surfaces of Wooden Barrels Are an Additional Microbial Inoculation Source for Lambic Beer Production

Abstract

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.

Keywords: amplicon sequencing; bacteria; culture-independent analysis; lambic beer; wooden barrels; yeasts.

FIG 1

(A) Wooden cask used for lambic beer production. (B) Bunghole used for sampling (now closed with a loose wooden panel). The specific features of the casks are given below the pictures. (C) Wooden foeder (F1 and F2) used for lambic beer production. (D) Wooden foeder (F3) used for lambic beer production. (Panels E) Manhole used for sampling. The specific features of the foeders are given below the pictures.

FIG 2

(A) Cleaning procedure for the wooden casks used for lambic beer production, between their emptying (removal of matured lambic beer) and filling with fresh wort. The different stages that were sampled are indicated as PC (precleaning), AC (after cleaning), AS (after sulfuring), and BF (before filling). (B) Cleaning procedure for the wooden foeders used for lambic beer production, between their emptying (removal of matured lambic beer) and filling with fresh wort. The different stages that were sampled are indicated as PC (precleaning) and AC (after cleaning).

FIG 3

Relative abundances of bacterial OTUs (A and C) and ASVs (B and D) obtained by amplicon sequencing of all inner wooden surfaces of casks (A and B) and foeders (C and D) used for lambic beer production, sampled during their respective cleaning procedures. OTUs with occurrences of <50 were discarded; all OTUs and ASVs with occurrences of <1,000 were grouped as “others.” In the case of the OTU-based analysis, the “others” group consisted of 66 different genera present at low abundances; in the case of the ASV-based analysis, the “others” group consisted of 64 different genera present at low abundances.

FIG 4

Relative abundances of fungal OTUs (A and C) and ASVs (B and D) obtained by amplicon sequencing of all inner wooden surfaces of casks (A and B) and foeders (C and D) used for lambic beer production, sampled during their respective cleaning procedures. OTUs with occurrences of <50 were discarded; all OTUs and ASVs with occurrences of <1,000 were grouped as “others.” The OTU Dipodascaceae was also grouped under “others,” because taxonomical classification yielded only the family level (representing 2,465 sequences). In the case of the OTU-based analysis, the “others” group consisted of 13 different genera present at low abundances; in the case of the ASV-based analysis, the “others” group consisted of 23 different genera present at low abundances.

FIG 5

Relative abundances of fungal ASVs, assigned to the species level, obtained by amplicon sequencing for all inner wooden surfaces of casks (A) and foeders (B) used for lambic beer production, sampled during their respective cleaning procedures. ASVs with occurrences of <1,000 were grouped as “others.”

FIG 6

PCoA biplots based on Bray-Curtis dissimilarity scores of the bacterial (A) and fungal (B) community structures of the inner surfaces of the wooden casks and foeders used for lambic beer production. Samples are categorized according to the different steps in the cleaning procedures. Foeders are circled in black.