Martin Sievers

Acetobacter europaeus sp. nov., a main component of industrial vinegar fermenters in central Europe

Summary A new species in the genus Acetobacter , for which we propose the name A. europaeus sp. nov., has been isolated and characterized in pure culture from high acid vinegar fermentations in Germany and Switzerland. In contrast to the other species of acetic acid bacteria, the isolated strains from industrial vinegar fermenters need acetic acid for growth. They could be cultivated on a special double layer agar with 4 to 8% acetic acid ( Entani et al., 1985). DNA-DNA hybridization between the DNA of 10 isolated A. europaeus strains as labelled DNA probe with the type strains of Acetobacter aceti, A. hansenii, A. liquefaciens, A. methanolicus, A. pasteurianus, A. xylinum, Gluconobacter oxydans and G. oxydans subsp. melanogenes indicated less than 25% DNA similarity. The important features of the new species are described. Acetobacter europaeus strain DES 11 (DSM 6160) is the type strain.

Acetobacter intermedius, sp. nov.

Strains of a new species in the genus Acetobacter, for which we propose the name A. intermedius sp. nov., were isolated and characterized in pure culture from different sources (Kombucha beverage, cider vinegar, spirit vinegar) and different countries (Switzerland, Slovenia). The isolated strains grow in media with 3% acetic acid and 3% ethanol as does A. europaeus, do, however, not require acetic acid for growth. These characteristics phenotypically position A. intermedius between A. europaeus and A. xylinus, DNA-DNA hybridizations of A. intermedius-DNA with DNA of the type strains of Acetobacter europaeus, A. xylinus, A. aceti, A. hansenii, A. liquefaciens, A. methanolicus, A. pasteurianus, A. diazotrophicus, Gluconobacter oxydans and Escherichia coli HB 101 indicated less than 60% DNA similarity. The important features of the new species are described. Acetobacter intermedius strain TF2 (DSM11804) isolated from the liquid phase of a tea fungus beverage (Kombucha) is the type strain.

Phylogenetic Positioning of Acetobacter, Gluconobacter, Rhodopila and Acidiphilium Species as a Branch of Acidophilic Bacteria in the α-subclass of Proteobacteria Based on 16S Ribosomal DNA Sequences

Summary 16S rRNA gene (rDNA) sequences from Acetobacter europaeus, Acetobacter xylinum, Acetobacter hansenii, Acetobacter liquefaciens, Acetobacter diazotrophicus, Acetobacter aceti, Acetobacter pasteurianus and Gluconobacter oxydans were determined. Phylogenetic trees were constructed that reflected the distant and close relationships. All investigated species had unique 16S rDNA sequences by demonstrated high sequence similarities (99.6 to 94.2%, corresponding to 7 to 83 base differences). Therefore, the studied species are closely related and represent together with other acidophilic bacteria ( Rhodopila globiformis and the Acidiphilium species) a distinct line of descent within the a-subclass of Proteobacteria .

Microbiology and fermentation balance in a kombucha beverage obtained from a tea fungus fermentation

Summary The transformation of sucrose into glucose, fructose, gluconic acid, ethanol, and acetic acid was determined during a 60 day tea fungus fermentation. Black tea containing 67.5 g sucrose per litre was inoculated with 10% fermentation broth including the cellulose containing coherent top layer of a previous tea fungus fermentation. The microflora embedded in the cellulose/acetan layer was characterized as a mixed culture of Acetobacter xylinum and Zygosaccbaromyces sp., respectively. The yeast cells converted sucrose into glucose and fructose. Fructose was metabolized prior to glucose. The pH value of the kombucha beverage decreased during fermentation from 3.75 to 2.42 as a result of acetic acid and gluconic acid formation. A fermentation balance of the substrates sucrose, glucose, fructose and products ethanol, acetic and gluconic acid and CO2 was calculated based on the carbon-mass (g substrate × number of C-atoms × 12 / molecular weight of substrate) as parameter. The total carbon-mass at the start of the fermentation was 30.5 g. The analogous values obtained after 10, 20, 30 and 40 days were 30.7 g, 30.5 g, 28.6 g, and 30.5, respectively. The good stoichiometry implies that all major fermentation products have been accounted for.

Phylogenetic Identification of Two Major Nitrogen-Fixing Bacteria Associated with Sugarcane

Acetobacter diazotrophicus and Herbaspirillum seropedicae were identified by genetic methods based on 16S rRNA sequences. A specific PCR method in combination with probing was developed for A. diazotrophicus. The PCR system includes four primers, of which the primers named AC (CTGTTTCCCGCAAGGGAC) and DI (GCGCCCCATTGCTGGGTT) generated an 445 bp amplicon in all of the 11 A. diazotrophicus strains tested. The phylogenetic position of H. seropedicae was determined. H. seropedicae forms with Oxalobacter formigenes a separate lineage in the beta-subclass of Proteobacteria.

Revival of the Species Acetobacter methanolicus (ex Uhlig et al. 1986) nom. rev.

Summary The 16S rRNA gene (rDNA) sequence from Acetobacter methanolicus MB58 was determined and aligned with known sequences of Acetobacter and Gluconobacter species from a previous study ( Sievers et al. 1994). Acetobacter methanolicus forms a single branch between Acetobacter pasteurianus / Acetobacter aceti and the cluster comprising Acetobacter europaeus, Acetobacter xylinum, Acetobacter hansenii, Acetobacter diazotrophicus and Acetobacter liquefaciens . The phylogenetic data provide sufficient and clear evidence for the revival of Acetobacter methanolicus from “ Acidomonas methanolica ”.

Characterization of the microflora of high acid submerged vinegar fermenters by distinct plasmid profiles

SummarySince isolation of the Acetobacter strains responsible for ethanol oxidation in high acid, submerged vinegar fermentations is still extremely difficult, characterization of the vinegar microflora was approached by isolation and gel electrophoresis of plasmids extractable from the harvested microorganisms. Distinct plasmid profiles have been detected in all of 19 investigated fermenters from 5 different locations. Plasmid sizes varied between 1.3 and 133 Megadalton. Between 3 and 11 different plasmids were recognized in particular profiles. Comparison of these profiles from different sources proves that the plasmid profile of a vinegar fermenter is a unique property of the inherent microflora which allows definite conclusions regarding its stability, origin, identity and composition.

Morphology, virulence and epidemiology of bacteriophage particles isolated from industrial vinegar fermentations

Summary Bacteriophage particles could be demonstrated in disturbed industrial vinegar fermentations both in submerged and trickling generators. A total of 300 samples from disturbed vinegar fermentations collected in Germany, Danmark and Austria, were examined by electron microscopy. 70% of the samples from submerged generators contained phage particles at numbers of at least 10 7 –10 9 /ml in the crude reactor content, whereas phage particles were always present in trickling generators at numbers of 10 6 –10 7 /ml. The phage particles were isometric-headed with long contractile tails, belonging to the group A of Bradley . 7 morphologically different types were identified based on head sizes, tail lengths and diameters. Head sizes varied between 60 and 110 nm. The corresponding tail lengths ranged from 99 to 360 nm with tail diameters from 14 to 31 nm. To prove infectivity of the observed phage particles for Acetobacter europaeus , the main species in industrial fermenters, a spirit vinegar culture in a laboratory submerged fermenter was infected with the bacteriophage fraction isolated by centrifugation from a trickling generator. The culture was lysed within a few hours, releasing high numbes of a specific bacteriophage as estimated by direct electron microscopy. This phage induced fermentation breakdown lasted for nearly 2 weeks. 18 days after fermentation stop, a new Acetobacter europaeus strain grew up in the fermenter characterized by a plasmid profile different from the strain present at the moment of phage addition. This experiment showed that the traditional trickling generator filled with beechwood chips is a potent source of virulent bacteriophages for cultures used in submerged fermenters. A. europaeus is not able to grow in soft agar preventing the use of the classical plaque test to enumerate infective bacteriophages.

Cloning, Sequence Analysis and Expression of the F1F0-ATPase β-Subunit from Wine Lactic Acid Bacteria

Summary The nucleotide sequences of the genes encoding the F 1 F 0 -ATPase β -subunit from Oenococcus oeni , Leuconostoc mesenteroides subsp. mesenteroides , Pediococcus damnosus , Pediococcus parvulus , Lactobacillus brevis and Lactobacillus hilgardii were determined. Their deduced amino acid sequences showed homology values of 79–98%. Data from the alignment and ATPase tree indicated that O. oeni and L. mesenteroides subsp. mesenteroides formed a group well-separated from P. damnosus and P. parvulus and from the group comprises L. brevis and L. hilgardii . The N-terminus of the F 1 F 0 -ATPase β -subunit of O. oeni contains a stretch of additional 38 amino acid residues. The catalytic site of the ATPase β -subunit of the investigated strains is characterized by the two conserved motifs GGAGVGKT and GERTRE. The amplified atpD coding sequences were inserted into the pCRT7/CT-TOPO vector using TA-cloning strategy and transformed in Escherichia coli . SDS-PAGE and Western blot analyses confirmed that O. oeni has an ATPase β -subunit protein which is larger in size than the corresponding molecules from the investigated strains.