Lambic sour beers are the products of a spontaneous fermentation that lasts for one to three years before bottling. The present study determined the microbiota involved in the fermentation of lambic beers by sampling two fermentation batches during two years in the most traditional lambic brewery of Belgium, using culture-dependent and culture-independent methods. From 14 samples per fermentation, over 2000 bacterial and yeast isolates were obtained and identified. Although minor variations in the microbiota between casks and batches and a considerable species diversity were found, a characteristic microbial succession was identified. This succession started with a dominance of Enterobacteriaceae in the first month, which were replaced at 2 months by Pediococcus damnosus and Saccharomyces spp., the latter being replaced by Dekkera bruxellensis at 6 months fermentation duration.

Estimation of the operational isolation units present in a MALDI-TOF mass spectral data set involves an essential dereplication step to identify redundant spectra in a rapid manner and without sacrificing biological resolution. We describe SPeDE, a new algorithm which facilitates culture-dependent clinical or environmental studies. SPeDE enables the rapid analysis and dereplication of isolates, a critical feature when long-term storage of cultures is limited or not feasible. We show that SPeDE can efficiently identify sets of similar spectra at the level of the species or strain, exceeding the taxonomic resolution of other methods. The high-throughput capacity, speed, and low cost of MALDI-TOF mass spectrometry and SPeDE dereplication over traditional gene marker-based sequencing approaches should facilitate adoption of the culturomics approach to bacterial isolation campaigns.

The effect of the growth medium used on the matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectra generated and its consequences for species and strain level differentiation of acetic acid bacteria (AAB) were determined by using a set of 25 strains. The strains were grown on five different culture media that yielded a total of more than 600 mass spectra, including technical and biological replicates. The results demonstrate that the culture medium can have a profound effect on the mass spectra of AAB as observed in the presence and varying signal intensities of peak classes, in particular when culture media do not sustain optimal growth. The observed growth medium effects do not disturb species level differentiation but strongly affect the potential for strain level differentiation. The data prove that a well-constructed and robust MALDI-TOF mass spectrometry identification database should comprise mass spectra of multiple reference strains per species grown on different culture media to facilitate species and strain level differentiation.

Three strains, LMG 27748^T, LMG 27749 and LMG 27882 with identical MALDI-TOF mass spectra were isolated from samples taken from the brewery environment. Analysis of the 16S rRNA gene sequence of strain LMG 27748^Trevealed that the taxon it represents was closely related to type strains of the species Gluconobacter albidus (100 % sequence similarity), Gluconobacter kondonii (99.9 %), Gluconobacter sphaericus (99.9 %) and Gluconobacter kanchanaburiensis (99.5 %). DNA–DNA hybridization experiments on the type strains of these species revealed moderate DNA relatedness values (39–65 %). The three strains usedd-fructose,d-sorbitol,meso-erythritol, glycerol,l-sorbose, ethanol (weakly), sucrose and raffinose as a sole carbon source for growth (weak growth on the latter two carbon sources was obtained for strains LMG 27748^Tand LMG 27882). The strains were unable to grow on glucose-yeast extract medium at 37 °C. They produced acid frommeso-erythritol and sucrose, but not from raffinose.d-Gluconic acid, 2-keto-d-gluconic acid and 5-keto-d-gluconic acid were produced fromd-glucose, but not 2,5-diketo-d-gluconic acid. These genotypic and phenotypic characteristics distinguish strains LMG 27748^T, LMG 27749 and LMG 27882 from species of the genus Gluconobacter with validly published names and, therefore, we propose classifying them formally as representatives of a novel species,Gluconobacter cerevisiaesp. nov., with LMG 27748^T( = DSM 27644^T) as the type strain.