Isolation of a lactic acid bacterium and yeast consortium from a fermented material of Ulva spp. (Chlorophyta)

AIMS: Microbiota in a fermented culture of Ulva spp. was examined with the objective to characterize the type of fermentation and to obtain starter microbes for performing seaweed fermentation. METHOD AND RESULTS: Fermented Ulva spp. cultures which were obtained and transferred in a laboratory were examined for their microbiota. With phenotypic characterization and phylogenetic analysis based on rRNA gene nucleotide sequences, the predominant micro-organisms were identified as Lactobacillus brevis, Debaryomyces hanseni var. hansenii, and a Candida zeylanoides-related specimen, suggesting that the observed fermentation can be categorized to lactic acid and ethanol fermentation. Inoculating the individually cultured cell suspensions of the three kinds of micro-organisms with cellulase induced the fermentation in various kinds of seaweed. CONCLUSIONS: A microbial consortium composed of a lactic acid bacterium, L. brevis, and yeasts, D. hansenii and a C. zeylanoides-related specimen, were predominant in a fermented culture of Ulva spp. Lactic acid and ethanol fermentation could be induced in various kinds of seaweed by adding this microbial consortium along with cellulase. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of lactic acid and ethanol fermentation in seaweed, which is expected to provide a new material for food and dietary applications.     

Comparative analysis of trehalose production by Debaryomyces hansenii and Saccharomyces cerevisiae under saline stress

The comparative analysis of growth, intracellular content of Na⁺ and K⁺, and the production of trehalose in the halophilic Debaryomyces hansenii and Saccharomyces cerevisiae were determined under saline stress. The yeast species were studied based on their ability to grow in the absence or presence of 0.6 or 1.0 M NaCl and KCl. D. hansenii strains grew better and accumulated more Na⁺ than S. cerevisiae under saline stress (0.6 and 1.0 M of NaCl), compared to S. cerevisiae strains under similar conditions. By two methods, we found that D. hansenii showed a higher production of trehalose, compared to S. cerevisiae; S. cerevisiae active dry yeast contained more trehalose than a regular commercial strain (S. cerevisiae La Azteca) under all conditions, except when the cells were grown in the presence of 1.0 M NaCl. In our experiments, it was found that D. hansenii accumulates more glycerol than trehalose under saline stress (2.0 and 3.0 M salts). However, under moderate NaCl stress, the cells accumulated more trehalose than glycerol. We suggest that the elevated production of trehalose in D. hansenii plays a role as reserve carbohydrate, as reported for other microorganisms.     

Debaryomyces hansenii strains with different cell sizes and surface physicochemical properties adhere differently to a solid agarose surface

The initial adhesion of four Debaryomyces hansenii strains to a solid agarose surface was investigated and correlated with their cell size and some cell surface physicochemical properties, i.e. (i) hydrophobicity and (ii) electron donor/acceptor ability. One strain adhered very poorly, whereas the three other strains were more adhesive. The former strain had a very hydrophilic cell surface, whereas the latter strains had more hydrophobic cell surfaces. In addition, the strain with the lowest adhesion among the adhesive strains had a more hydrophobic cell surface than the two most adhesive strains. Finally, the more adhesive the strain was, the larger it was, and the better it was to donate electrons from its cell surface. These results show a clear relationship between the cell size, the cell surface physicochemical properties, and the initial adhesion of D. hansenii. A possible explanation of this relationship is discussed.     

Genes from Debaryomyces hansenii increase salt tolerance in Saccharomyces cerevisiae W303

The yeast Debaryomyces hansenii has been chosen as a model for molecular studies of tolerance to NaCl. A gene library was built and transformants of Saccharomyces cerevisiae W303 containing genes from D. hansenii were selected for their ability to grow in the presence of high concentrations of NaCl and/or low concentrations of KCl. In three of these transformants 500 mM NaCl improved growth at pH 7.6 like in D. hansenii but not in S. cerevisiae. One of the plasmids restored growth at 50 microM KCl and K(+) uptake in a mutant of S. cerevisiae lacking genes that encode K(+) transporters.     

Debaryomyces udenii,sp. nov. (Saccharomycetaceae), a new species from soil

A new, soil-associated species of the genus Debaryomyces, D. udenii, is described. The species is characterized by pusticulate rather than verrucate ascospores, and slowly lytic asci.     

On the mechanism of salt tolerance

As glycerol was suggested as an osmotic agent in the salt tolerantDebaryomyces hansenii the concentrations of total, intracellular, and extracellular glycerol produced by this yeast was followed during growth in 4 mM, 0.68 M, and 2.7 M NaCl media. The total amount of glycerol was not directly proportional to biomass production but to the cultural salinity with maximum concentrations just prior to or at the beginning of the stationary phase. In all cultures the cells lost some glycerol to the media, at 2.7 M NaCl the extracellular glycerol even amounted maximally to 80% of the total. A distinct maximum of intracellular glycerol, related to dry weight or cell number, appeared during the log phase at all NaCl concentrations. As the intracellular calculated glycerol concentrations amounted to 0.2 M, 0.8 M, and 2.6 M in late log phase cells at 4 mM, 0.68 M, and 2.7 M NaCl, respectively, whereas the corresponding analysed values for the glycerol concentrations of the media were 0.7 mM, 2.5 mM, and 3.0 mM, glycerol contributes to the osmotic balance of the cells.During the course of growth all cultures showed a decreasing heat production related to cell substance produced, most pronounced at 2.7 M NaCl. At 2.7 M NaCl the total heat production amounted to-1690 kJ per mole glucose consumed in contrast to-1200 and-1130 kJ at 4 mM and 0.68 M NaCl, respectively. TheY _m -values were of an inverse order, being 129, 120, and 93 at 4 mM, 0.68 M, and 2.7 M NaCl, respectively.     

Efficient one-step starch utilization by industrial strains of Saccharomyces cerevisiae expressing the glucoamylase and α-amylase genes from Debaryomyces occidentalis

Amylolytic industrial polyploid strains of Saccharomyces cerevisiae (ATCC 4126, ATCC 9763 and ATCC 24858) expressing a glucoamylase gene (GAM1) or an α-amylase gene (AMY) from Debaryomyces occidentalis were developed. The glucoamylase activity of S. cerevisiae ATCC 9763 expressing the GAM1 gene was 3.7-times higher than that of D. occidentalis. On the other hand, α-amylase activity in the corresponding strain expressing the D. occidentalis AMY gene increased 10-times relative to D. occidentalis. These two recombinant yeast strains expressing the GAM1 gene and AMY gene, respectively were cultured simultaneously to produce both glucoamylase and α-amylase for efficient one-step utilization of starch. Growth, substrate utilization and enzyme activity of these strains are described.     

Purification,characterization, and potential applications of formate oxidase from <Emphasis Type="Italic">Debaryomyces vanrijiae</Emphasis> MH201

Formate oxidase was found in cell-free extracts of Debaryomyces vanrijiae MH201, a soil isolate. After purification by column chromatography, the preparation showed a protein band corresponding to a molecular mass (MM) of 64 kDa on sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The MM, estimated by a gel filtration, was 99 kDa. The preparation showed two and three bands on isoelectric focusing under denaturing and native conditions, respectively. These results suggest that the preparation contained three isoforms, each of which might be composed of αα, αβ, and ββ subunits with apparently similar MM. The preparation acted on formate with K _m and V _max values of 11.7 mM and 262 μmol min⁻¹ mg⁻¹, respectively, at pH 4.5 and 25°C, but showed no evidence of activity on the other compounds tested. The optimum pH and temperature were pH 4.0 and 35°C, respectively. The preparation showed activities of 85% of the initial activity after storage at pH 6.0 and 4°C for 8 weeks. When 10 mM formaldehyde was reacted with 2.0 U ml⁻¹ of the enzyme preparation at pH 5.5 and room temperature in the presence of 2.0 U ml⁻¹ of a microbial aldehyde oxidase and 100 U ml⁻¹ of catalase for 180 min, neither of formate nor formaldehyde was detected, suggesting that the reaction involved the quantitative conversion of formaldehyde to carbon dioxide.     

Phenotypic and genotypic identification of yeasts from cheese

Eighty-five yeast strains isolated from different cheeses of Austria, Denmark, France, Germany, and Italy were identified using physiological methods and genotypically using random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) analysis. Good congruence was found between the phenotypic and genotypic data for 39 of the isolates. However, 26 isolates of Geotrichum could only be identified to the species level using the genotypic methods and 7 isolates were correctly identified to the genus level only using phenotypic identification methods. The phenotypic identification did not agree with the genotypic data for 14 yeast isolates. Using ubiquinone analysis, yeast cell wall sugars and the diazonium blue B test 5 incorrectly identified isolates with phenotypic methods could be identified genotypically. In addition the 7 isolates identified only to the genus level by the phenotypic methods and the 26 Geotrichum strains were identified to the species level using the polyphasic molecular approach mentioned above. Eleven strains remained unidentified. The 76 identified yeast isolates were assigned to 39 species, the most frequent assignments were made to Debaryomyces hansenii, Geotrichum candidum, Issatchenkia orientalis, Kluyveromyces lactis, K. marxianus, Saccharomyces cerevisiae, Yarrowia lipolytica, andCandida catenulata. It is proposed that Debaryomyces hansenii (Zopf) Lodder et Kreger-van Rij and Debaryomyces fabryi Ota should be reinstated. The RAPD-PCR data reinforced the view that the species Galactomyces geotrichum is heterogeneous with all of the Geotrichum isolates from cheese products being assigned G. geotrichum group A sensu M.T. Smith. It is suggested that the name Geotrichum candidum be conserved for this rather common species.     

Activation of fermentation by salts in Debaryomyces hansenii

The presence of 1.0 M KCl or NaCl during growth of Debaryomyces hansenii results in increased ethanol production. An additional increase of fermentation was observed when the salts were also present during incubation under nongrowing conditions. Extracts of cells grown in the presence of salt showed increased alcohol dehydrogenase and phosphofructokinase activities, indicating that these enzymes are responsible for the increased fermentation capacity. This is confirmed by measurements of the glycolytic intermediates. The increased fermentation capacity of the cells grown with salts seems to enable them to cope with the additional energy required for uptake and/or efflux of cations.