Whole cells are usually used for oxygenase-catalysed biotransformations to ensure efficient cofactor recycling and to avoid problems with purification and stability of these complex, multi-subunit enzymes. Productivity in oxygenase-catalysed biotransformations is frequently restricted by toxicity of the substrates to the cells, but attempting to solve this problem using two-liquid phase reaction systems with conventional solvents provides only modest improvements in productivity. Therefore, we developed new, two phase systems using water-immiscible ionic liquids. Even though [NMeOct 3 ][NTf 2 ] and [P 6,6,6,14 ][NTf 2 ], inhibited growth of Escherichia coli by 39% and 23% respectively, both ionic liquids could be used to improve toluene dioxygenase-catalysed conversion of toluene to toluene cis-glycol using recombinant E. coli cells. The two-liquid phase reaction system improved resistance to toluene toxicity by 8-fold, and product concentrations increased by 2.5-fold in shake flask cultures. Product concentrations and specific product yields were improved by 200% and 238% respectively in bioreactors with an unrestricted oxygen supply.
The human foot provides an ideal environment for the colonization and growth of bacteria and subsequently is a body site associated with the liberation of odour. This study aimed to enumerate and spatially map bacterial populations' resident across the foot to understand any association with odour production. Culture-based analysis confirmed that Staphylococci were present in higher numbers than aerobic corynebacteria and Gram-positive aerobic cocci, with all species being present at much higher levels on the plantar sites compared to dorsal sites. Microbiomic analysis supported these findings demonstrating that Staphylococcus spp. were dominant across different foot sites and comprised almost the entire bacterial population on the plantar surface. The levels of volatile fatty acids, including the key foot odour compound isovaleric acid, that contribute to foot odour were significantly increased at the plantar skin site compared to the dorsal surface. The fact that isovaleric acid was not detected on the dorsal surface but was present on the plantar surface is probably attributable to the high numbers of Staphylococcus spp. residing at this site. Variations in the spatial distribution of these microbes appear to be responsible for the localized production of odour across the foot.
Fourier transform infrared (FT-IR) spectroscopy was employed as a rapid high-throughput phenotypic typing technique to generate metabolic fingerprints of Escherichia coli MG1655 pDTG601A growing in fed-batch culture, during the dioxygenase-catalysed biotransformation of toluene to toluene cis-glycol. With toluene fed as a vapour, the final toluene cis-glycol concentration was 83 mM, whereas the product concentration was only 22 mM when the culture was supplied with liquid toluene. Multivariate statistical analysis employing cluster analysis was used to analyse the dynamic changes in the data. The analysis revealed distinct trends and trajectories in cluster ordination space, illustrating phenotypic changes related to differences in the growth and product formation of the cultures. In addition, partial least squares regression was used to correlate the FT-IR metabolic fingerprints with the levels of toluene cis-glycol and acetate, the latter being an indicator of metabolic stress. We propose that this high-throughput metabolic fingerprinting approach is an ideal tool to assess temporal biochemical dynamics in complex biological processes, as demonstrated by this redox biotransformation. Moreover, this approach can also give useful information on product yields and fermentation health indicators directly from the fermentation broth without the need for lengthy chromatographic analysis of the products.
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