Proposal of Thorsellia kenyensis sp. nov. and Thorsellia kandunguensis sp. nov., isolated from larvae of Anopheles arabiensis, as members of the family Thorselliaceae fam. nov.

Two Gram-negative, rod-shaped strains, T2.1(T) and W5.1.1(T), isolated from larvae of the mosquito Anopheles arabiensis, were investigated using a polyphasic approach. On the basis of 16S rRNA gene sequence similarity studies, strains T2.1(T) and W5.1.1(T) were shown to belong to the genus Thorsellia, both showing 97.8 % similarity to the type strain of Thorsellia anophelis, with 98.1 % similarity to each other. Chemotaxonomic data supported the allocation of the strains to the genus Thorsellia: their major fatty acids were C18 : 1ω7c, C16 : 0 and C14 : 0 and they harboured a ubiquinone Q-8 quinone system and a polyamine pattern with the major compound 1,3-diaminopropane. Qualitative and quantitative differences in their polar lipid profiles distinguished strains T2.1(T) and W5.1.1(T) from each other and from T. anophelis. Average nucleotide identity (ANI), DNA-DNA hybridization, multilocus sequence analysis (MLSA) as well as physiological and biochemical tests allowed T2.1(T) and W5.1.1(T) to be distinguished both genotypically and phenotypically from each other and from the type strain of T. anophelis. Thus, we propose that these isolates represent two novel species of the genus Thorsellia, named Thorsellia kenyensis sp. nov. (type strain T2.1(T) = CCM 8545(T) = LMG 28483(T) = CIP 110829(T)) and Thorsellia kandunguensis sp. nov. (type strain W5.1.1(T) = LMG 28213(T) = CIP 110794(T)). Furthermore, phylogenetic analysis based on nearly full-length 16S rRNA gene sequences showed that the genus Thorsellia forms a separate branch, distinct from the families Enterobacteriaceae, Pasteurellaceae and Orbaceae. As a consequence, a new family Thorselliaceae fam. nov. is proposed. An emended description of Thorsellia anophelis is also provided.

Interaction of Lactobacillus vini with the ethanol-producing yeasts Dekkera bruxellensis and Saccharomyces cerevisiae.

Lactobacillus vini was recently described as a contaminant in industrial ethanol fermentations and its co-occurrence with Dekkera bruxellensis was noted. We investigated the growth characteristics of L. vini in cocultivation together with either Saccharomyces cerevisiae or D. bruxellensis. Lower cell numbers of both the yeasts and L. vini as well as a decrease in ethanol and lactate formation in mixed batch cultures compared with pure cultures were noted. L. vini formed cell aggregates (flocs) in all cultivation media with different shapes in Man-Rogosa-Sharpe and yeast extract-peptone-dextrose media. Flocs' size and proportion of cells bound to flocs increased with increasing ethanol concentration. In coculture, formation of lactic acid bacteria-yeast cell aggregates consisting of a bacterial core with an outer layer of yeast cells was observed. L. vini-D. bruxellensis flocs had a bigger surface, due to cells protruding from the pseudomycelium. The involvement of mannose residues in the flocculation between L. vini and yeasts was tested. The presence of mannose induced deflocculation in a concentration-dependent manner. Less mannose was required for the deflocculation of D. bruxellensis as compared with S. cerevisiae.

Fermentation characteristics of Dekkera bruxellensis strains.

The influence of pH, temperature and carbon source (glucose and maltose) on growth rate and ethanol yield of Dekkera bruxellensis was investigated using a full-factorial design. Growth rate and ethanol yield were lower on maltose than on glucose. In controlled oxygen-limited batch cultivations, the ethanol yield of the different combinations varied from 0.42 to 0.45 g (g glucose)(-1) and growth rates varied from 0.037 to 0.050 h(-1). The effect of temperature on growth rate and ethanol yield was negligible. It was not possible to model neither growth rate nor ethanol yield from the full-factorial design, as only marginal differences were observed in the conditions tested. When comparing three D. bruxellensis strains and two industrial isolates of Saccharomyces cerevisiae, S. cerevisiae grew five times faster, but the ethanol yields were 0-13% lower. The glycerol yields of S. cerevisiae strains were up to six-fold higher compared to D. bruxellensis, and the biomass yields reached only 72-84% of D. bruxellensis. Our results demonstrate that D. bruxellensis is robust to large changes in pH and temperature and may have a more energy-efficient metabolism under oxygen limitation than S. cerevisiae.

Interaction of vitronectin with Haemophilus influenzae.

Eight strains of Haemophilus influenzae were tested for binding to human vitronectin. All strains adhered to vitronectin-coated glass slides but no binding was detected using soluble vitronectin, suggesting that surface association of vitronectin is a prerequisite. Vitronectin binding was not likely to be mediated by fimbriae as non-fimbriated and fimbriated isogenic strains adhered equally. Adhesion could be blocked by heparin, which is also known to block vitronectin binding to Staphylococcus aureus. However, no blocking was achieved with sialic acid-rich glycoproteins such as fetuin and mucin contrasting with Helicobacter pylori for which sialic acid seems to play an important role. With Streptococcus pneumoniae binding was detected both with soluble and surface-associated vitronectin and could not be blocked by heparin. Our results suggest that H. influenzae, Streptococcus pneumoniae and Helicobacter pylori all use distinct modes to interact with vitronectin.

Transcriptome of the alternative ethanol production strain Dekkera bruxellensis CBS 11270 in sugar limited, low oxygen cultivation.

Dekkera bruxellensis can outcompete Saccharomyces cerevisiae in environments with low sugar concentrations. It is usually regarded as a spoilage yeast but has lately been identified as an alternative ethanol production organism. In this study, global gene expression in the industrial isolate D. bruxellensis CBS 11270 under oxygen and glucose limitation was investigated by whole transcriptome sequencing using the AB SOLiD technology. Among other observations, we noted expression of respiratory complex I NADH-ubiquinone reductase although D. bruxellensis is a Crabtree positive yeast. The observed higher expression of NADH-generating enzymes compared to NAD(+)-generating enzymes might be the reason for the previously observed NADH imbalance and resulting Custer effect in D. bruxellensis. Low expression of genes involved in glycerol production is probably the molecular basis for high efficiency of D. bruxellensis metabolism under nutrient limitation. No D. bruxellensis homologs to the genes involved in the final reactions of glycerol biosynthesis were detected. A high number of expressed sugar transporter genes is consistent with the hypothesis that the competitiveness of D. bruxellensis is due to a higher affinity for the limiting substrate.

High density process to cultivate Lactobacillus plantarum biomass using wheat stillage and sugar beet molasses

Background: Owing to the growing interest in biofuels, the concept of a biorefinery where biomass is converted to a variety of useful products is gaining ground. We here present how distillery waste is combined with a by-product from a sugar production, molasses, to form a medium for the growth of Lactobacillus plantarum with yields and biomass densities comparable with conventional industrial media. Such approach enables a cost-effective utilization of the problematic wastewater from ethanol and a by-product from sugar production. It is the first approach that attempts to find low-cost media for the production of Lactobacillus plantarum biomass. Results: This study suggests that sieved wheat stillage enriched by adding 1.77 g/l yeast extract and 10 percent molasses (v/v), with NH4OH used for pH adjustment, may be used as a media for large-scale cultivation of L. plantarum. Such composition of the medium permits a high density of lactic acid bacteria (1.6 x 10(10) cfu/ml) to be achieved. Conclusions: The use of a fermentation medium consisting of distillery wastewater and molasses to obtain value-added products (such as LAB biomass and lactic acid) is a possible step for classical ethanol production to move towards a biorefinery model production in which all by and waste products are utilized to increase produced values and reduce waste production. This enables a cost-effective utilization of the problematic wastewater from ethanol and sugar production.