The same happened in the cases of A. pushchinoensis, A. kestanbolensis, A. eryuanensis and A. tengchongensis. Although no discernible increase in turbidity (OD600 nm) was measured at concentrations of ethanol in the media above 10%, a biofilm consisting of bacterial cells enclosed in an extracellular polysaccharide matrix actively growing on a surface (Hamon & Lazazzera, 2001) was observed on the glass surface of the bottles after a 24-h incubation. Moreover, multilayer biofilms were clearly seen even though the ethanol concentration eventually reached 13% at 60 °C (Fig. 2a). The Roxadustat ic50 freely suspended cells of strain E13T incubated with

8% ethanol showed a tendency to aggregate. Some cells adhered to each other and formed tree-like structures, which might be important for its initial attachment to a surface (Fig. 2b). Biofilm formation

is an important strategy for bacterial accumulation in natural aquatic habitats. Biofilms have been proposed to constitute an environmental refuge for a number of bacteria and to provide bacteria with an adaptive advantage promoting their environmental persistence (Matz et al., 2005). In many bacteria, especially strains of pathogenic genus, ethanol stress has been reported to lead to induction INK 128 concentration of biofilm formation (Knobloch et al., 2006; Mukherjee et al., 2006). Therefore, we suggest that the biofilm formation by strain E13T has an important contribution to the adaptive advantage of growth under high ethanol stress conditions. The ability of A. flavithermus CM to

produce biofilms has been investigated (Burgess et al., 2009). The biofilm of A. flavithermus DSM 2641T was also observed in LB medium without ethanol after 12 h of incubation at 60 °C. The cells of strain E13T appeared as Gram-staining-positive, motile, spore-forming rods. At stationary phase, the cells were 0.4–0.7 μm in width and 1.2–7.0 μm in length. The temperature growth range was from 30 to 66 °C with an optimal growth at 60 °C. The pH growth range was from 5.5 to 10.0 with an optimum growth at 7.0–7.5. The strain E13T was catalase positive while it was negative for gelatin hydrolysis, starch hydrolysis, nitrate reduction, check indole production and phenylalanine deaminase. Growth of strain E13T was inhibited in the presence of NaCl concentration above 3.5% (w/v) and the optimal NaCl concentration for growth was 0.3% (w/v). The isolate E13T utilized a wide range of carbon sources including arabinose, cellobiose, galactose, gluconate, glucose, maltose, mannitol, sucrose, trehalose and xylose. The following carbon sources did not support growth: ethanol, fructose, lactose, mannose, rhamnose and ribose. The differentiating phenotypic features between the new isolate and phylogenetically as well as phenotypically related species are indicated in Table 1. The major distinctions include substrate specificities with particularly good growth on arabinose and xylose and the lack of growth on mannose.