Sulfate (SO) is an often-utilized and well-understood inorganic sulfur source in microorganism culture. Recently, another inorganic sulfur source, thiosulfate (SO), was proposed to be more advantageous in microbial growth and biotechnological applications. Although its assimilation pathway is known to depend on O-acetyl-L-serine sulfhydrylase B (CysM in Escherichia coli), its metabolism has not been extensively investigated. Therefore, we aimed to explore another yet-unidentified CysM-independent thiosulfate assimilation pathway in E. coli. ΔcysM cells could accumulate essential L-cysteine from thiosulfate as the sole sulfur source and could grow, albeit slowly, demonstrating that a CysM-independent thiosulfate assimilation pathway is present in E. coli. This pathway is expected to consist of the initial part of the thiosulfate to sulfite (SO) conversion, and the latter part might be shared with the final part of the known sulfate assimilation pathway [sulfite → sulfide (S) → L-cysteine]. This is because thiosulfate-grown ΔcysM cells could accumulate a level of sulfite and sulfide equivalent to that of wild-type cells. The catalysis of thiosulfate to sulfite is at least partly mediated by thiosulfate sulfurtransferase (GlpE), because its overexpression could enhance cellular thiosulfate sulfurtransferase activity in vitro and complement the slow-growth phenotype of thiosulfate-grown ΔcysM cells in vivo. GlpE is therefore concluded to function in the novel CysM-independent thiosulfate assimilation pathway by catalyzing thiosulfate to sulfite. We applied this insight to L-cysteine overproduction in E. coli and succeeded in enhancing it by GlpE overexpression in media containing glucose or glycerol as the main carbon source, by up to ~1.7-fold (1207 mg/l) or ~1.5-fold (1529 mg/l), respectively.

In the startup of a polymer electrolyte fuel cell below subzero temperature, one problem is freezing of the produced water, inducing operation shutdown and various kinds of degradation of the cell performance. To evaluate the freezing behavior during rising temperature, this study conducted measurement of cold start characteristics under simulated adiabatic conditions and observation of ice distribution in the cell by a cryo-SEM. The results suggested different ice distributions depending on the timing of supercooling release, and a large influence of the ice layer formed at the interface between the catalyst layer and the MPL on the cold stat characteristics. A hydrophilic MPL was applied to improve the startup ability, and compared with the conventional hydrophobic MPL. It was shown that the hydrophilic MPL suppresses the formation of ice layer at the interface and successfully improves the startup ability, indicating a suitable structure for cold startup.

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