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 ( species more than species) proliferated during two phases of the lambic beer production process, represented by during a few days in the beginning of the fermentation and 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. 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, at the beginning and in a later stage of the production process. This developed framework could also be applied for other fermentation processes.

Microbial acidification, alcoholization, and aroma production during spontaneous lambic beer production

“…The microbiology of the lambic beer production process has been described as a succession of four phases, based on culture‐dependent microbiological analyses (Fig. ): (i) an enterobacterial and wild (oxidative) yeast phase with a limited growth of AAB; (ii) an alcoholic or main fermentation phase with S. cerevisiae and/or S. pastorianus ; (iii) an acidification phase with P. damnosus and/or Lactobacillus brevis as well as A. pasteurianus ; and (iv) a maturation phase with P. damnosus and B. bruxellensis . American coolship ale productions display similar microbial profiles, as revealed by amplicon sequencing targeting both bacterial and yeast species and showing the presence of Acetobacter species .…”

“…Moreover, AAB are regarded as undesirable spoilers of alcoholic fermentations (wine, cider, and beer). However, they do play a functional role in cocoa bean, kombucha, and lambic beer fermentations . Although their growth is optimal at 25–30 °C and in the presence of 0.35% acetic acid for almost all genera, the sporadic isolation of Acetobacter and Gluconobacter species throughout traditional lambic beer fermentation and maturation processes reflects possible difficulties for their growth and recovery, probably due to the VBNC state of their cells .…”

“…Moreover, two new AAB species have been described that seem to be characteristic for acidic beers, namely Acetobacter lambici and Gluconobacter cerevisiae . It has been shown that the obligate aerobic AAB are concentrated at the wort/air interface of the barrels and, hence, may be under‐represented through submerged sampling . Further, these investigations have shown the presence of Acetobacter orientalis during a few days in the beginning of the fermentation and A. pasteurianus from seven weeks until 24 months of maturation .…”

“…It has been shown that the obligate aerobic AAB are concentrated at the wort/air interface of the barrels and, hence, may be under‐represented through submerged sampling . Further, these investigations have shown the presence of Acetobacter orientalis during a few days in the beginning of the fermentation and A. pasteurianus from seven weeks until 24 months of maturation . Competitive exclusion tests combined with comparative genomic analysis of all genomes of strains of both species available has disclosed possible reasons for this successive dominance, in particular reflecting adaptation toward high concentrations of ethanol and increasing concentrations of acetic acid .…”

“…Further, spatial analysis has revealed that significantly higher concentrations of acetic acid (from ethanol) and acetoin (from lactic acid) are produced at the air/liquid interface in the casks, due to higher AAB counts and higher metabolic activity of the AAB species during the first six months of lambic beer production. In contrast, no differences in AAB species diversity occur throughout the fermenting wort in the casks . Similarly, A. pasteurianus has been isolated from Belgian red‐brown acidic ale productions, whose maturation takes place in vertical wooden barrels with headspace …”