The Norwegian brewery, which also produces cider from its own orchard, prepared 1100 liters of wort from malted barley, 30 percent unmalted wheat, and aged dry hops, acidified to pH 4.9 with lactic acid in imitation of Belgian practice. The wort cooled overnight in a metal coolship open to the ambient air, and two hours before the end of cooling the brewers added 40 kilograms of freshly harvested whole Aroma apples. The cooled wort was then split into three similar 225-liter oak barrels that had previously held American Merlot wine and a plum beer, cleaned with warm water at 100 bar pressure before filling. These barrels, sampled repeatedly from two hours to 27 months after filling, served as biological triplicates for the study.

The analytical arsenal deployed was formidable. Selective agar media allowed researchers to count presumptive lactic acid bacteria, acetic acid bacteria, yeasts, and enterobacteria. Amplicon sequencing of the bacterial 16S rRNA gene and the fungal ITS1 region revealed which genera were present, while shotgun metagenomic sequencing of whole-community DNA, yielding more than 134 million high-quality reads and 41.7 billion base pairs, enabled species-level identification through fragment recruitment plotting. Cell-free supernatants were analyzed for carbohydrates, sugar alcohols, organic acids, amino acids, biogenic amines, and dozens of volatile organic compounds, giving a parallel picture of the chemistry unfolding inside each barrel.

The results confirmed that the same microbial succession known from Belgian lambic breweries took place in Norway, but with blurred boundaries. In classic lambic production, four phases follow one another: an initial fermentation dominated by enterobacteria and non-Saccharomyces yeasts, a main fermentation driven by Saccharomyces, an acidification phase led by Pediococcus and acetic acid bacteria, and a long maturation dominated by Brettanomyces. In the Norwegian barrels, these phases occurred but overlapped considerably. In barrel 2, for example, Brettanomyces species appeared as early as day 27, coinciding with Saccharomyces and enterobacteria, so that main fermentation, acidification, and maturation ran simultaneously.

The early days of fermentation were dominated by enterobacteria, reaching plate counts as high as log 9.6 colony-forming units per milliliter despite the manual acidification of the wort, which was clearly insufficient to suppress them. Shotgun metagenomics identified Rahnella variigena, an unidentified Pseudescherichia species close to Pseudescherichia vulneris, an unidentified Leclercia species, and a Klebsiella species related to Klebsiella huaxiensis. Remarkably, Rahnella variigena has never before been identified in beer. Its varying abundance across the three barrels correlated with concentrations of dimethyl sulfide, acetoin, and 2,3-butanediol, compounds that enterobacteria produce from monosaccharides and that can impart off-flavors. The cadaverine detected in all barrels was plausibly linked to the Klebsiella species, which carries a lysine decarboxylase gene.

Acetic acid bacteria made an early but barrel-dependent appearance. Acetobacter cerevisiae dominated barrel 2 in the first hours, reaching 59.7 percent relative abundance, while barrel 3 hosted Gluconobacter cerinus at 44.6 percent. Barrel 1, filled first from the bottom of the coolship, contained no acetic acid bacteria at all, likely because these aerobic microbes congregated at the liquid-air interface during cooling. Intriguingly, both A. cerevisiae and G. cerinus are known from apple surfaces and cider fermentations, hinting that the added apples may have contributed them, although the absence of an apple-free control fermentation means the researchers cannot be certain. The apple addition did visibly boost fruit-surface microbes such as Metschnikowia, Aureobasidium, and Sporobolomyces at the coolship stage, but only the bacterial family Erwiniaceae, including Pantoea agglomerans and Erwinia billingiae, persisted beyond it.

The main fermentation revealed a surprise: two different Saccharomyces species appeared in different barrels. Saccharomyces bayanus emerged early in barrels 1 and 3, possibly because of its greater tolerance of the cellar’s cool temperatures, while S. cerevisiae peaked at month 6 in barrel 2. Since the brewery also ferments cider spontaneously, and S. bayanus is a common cider yeast, the wood of the barrels may have harbored it. The Saccharomyces yeasts consumed fructose, glucose, and sucrose within three days, then maltose by month 2 or 12 depending on the barrel, producing ethanol concentrations near 50 grams per liter along with higher alcohols and esters that shape the beer’s aroma.

The acidification and maturation phases then converged on the familiar lambic key players. Pediococcus damnosus, the great acidifier of lambic beer, appeared from month 6 or 12 onward and reached nearly half of the microbial community in barrel 3, driving the pH down from 4.9 to about 3.6 and pushing total lactic acid to roughly 5.7 to 6.0 grams per liter in an almost equimolar mix of the L and D forms. Brettanomyces species, chiefly B. custersianus, B. bruxellensis, and B. anomalus, degraded the leftover maltooligosaccharides and generated the signature volatile phenols 4-ethylphenol and 4-ethylguaiacol, which appeared within the first month at constant levels of about 1 to 2 milligrams per liter. Ethyl acetate and ethyl lactate rose in two waves, one coinciding with Saccharomyces and the later one with Brettanomyces, while acetoin produced by P. damnosus was converted into 2,3-butanediol by the Brettanomyces yeasts as part of their redox balancing.

Perhaps the most sobering finding is that the three barrels, filled with identical wort and stored side by side in the same cellar, developed measurably different microbial communities and metabolite profiles, underscoring that each wooden barrel acts as its own reservoir of resident microbes and its own micro-environment. The researchers caution that without an apple-free control, their conclusions apply only to this specific apple batch from this specific orchard, and that batch-to-batch variability at the brewery remains unexplored. Still, the message is clear: the essence of lambic, a self-organizing community of enterobacteria, Saccharomyces, Pediococcus, and Brettanomyces orchestrated by wooden barrels rather than by any magical valley air, can be recreated far from Brussels. The Senne valley may hold the tradition, but the microbes, it turns out, are willing travelers.

Subject of Research: Microbial ecology and metabolite dynamics of spontaneously fermented lambic-style acidic beer produced in Norway

Article Title: Norwegian lambic-style acidic beers share key microorganisms and metabolites with Belgian lambic beers, although typical microbial phases are less pronounced

Article References: Vermote, L., Jenkins, A., Moen, B., Axelsson, L., De Vuyst, L., & Weckx, S. (2026). Norwegian lambic-style acidic beers share key microorganisms and metabolites with Belgian lambic beers, although typical microbial phases are less pronounced. Heliyon, 12(15), Article e45521. https://doi.org/10.1016/j.heliyon.2026.e45521

Image Credits: AI Generated

DOI: 10.1016/j.heliyon.2026.e45521

Keywords: lambic beer, spontaneous fermentation, sour beer, Brettanomyces, Pediococcus damnosus, Saccharomyces, enterobacteria, acetic acid bacteria, shotgun metagenomics, wooden barrels, apple addition, food microbiology