The worldwide surplus of glycerol generated as inevitable byproduct of biodiesel fuel and oleochemical production is resulting in the shutdown of traditional glycerol-producing/refining plants and new applications are needed for this now abundant carbon source. In this article we report our finding that Escherichia coli can ferment glycerol in a pH-dependent manner. We hypothesize that glycerol fermentation is linked to the availability of CO 2 , which under acidic conditions is produced by the oxidation of formate by the enzyme formate hydrogen lyase (FHL). In agreement with this hypothesis, glycerol fermentation was severely impaired by blocking the activity of FHL. We demonstrated that, unlike CO 2 , hydrogen (the other product of FHL-mediated formate oxidation) had a negative impact on cell growth and glycerol fermentation. In addition, supplementation of the medium with CO 2 partially restored the ability of an FHL-deficient strain to ferment glycerol. High pH resulted in low CO 2 generation (low activity of FHL) and availability (most CO 2 is converted to bicarbonate), and consequently very inefficient fermentation of glycerol. Most of the fermented glycerol was recovered in the reduced compounds ethanol and succinate (93% of the product mixture), which reflects the highly reduced state of glycerol and confirms the fermentative nature of this process. Since glycerol is a cheap, abundant, and highly reduced carbon source, our findings should enable the development of an E. coli-based platform for the anaerobic production of reduced chemicals from glycerol at yields higher than those obtained from common sugars, such as glucose. ß

Availability, low prices, and a high degree of reduction make glycerol an ideal feedstock to produce reduced chemicals and fuels via anaerobic fermentation. Although glycerol metabolism in Escherichia coli had been thought to be restricted to respiratory conditions, we report here the utilization of this carbon source in the absence of electron acceptors. Cells grew fermentatively on glycerol and exhibited exponential growth at a maximum specific growth rate of 0.040 ؎ 0.003 h ؊1 . The fermentative nature of glycerol metabolism was demonstrated through studies in which cell growth and glycerol utilization were observed despite blocking several respiratory processes. The incorporation of glycerol in cellular biomass was also investigated via nuclear magnetic resonance analysis of cultures in which either 50% U-13 C-labeled or 100% unlabeled glycerol was used. These studies demonstrated that about 20% of the carbon incorporated into the protein fraction of biomass originated from glycerol. The use of U-13 C-labeled glycerol also allowed the unambiguous identification of ethanol and succinic, acetic, and formic acids as the products of glycerol fermentation. The synthesis of ethanol was identified as a metabolic determinant of glycerol fermentation; this pathway fulfills energy requirements by generating, in a redox-balanced manner, 1 mol of ATP per mol of glycerol converted to ethanol. A fermentation balance analysis revealed an excellent closure of both carbon (ϳ95%) and redox (ϳ96%) balances. On the other hand, cultivation conditions that prevent H 2 accumulation were shown to be an environmental determinant of glycerol fermentation. The negative effect of H 2 is related to its metabolic recycling, which in turn generates an unfavorable internal redox state. The implications of our findings for the production of reduced chemicals and fuels were illustrated by coproducing ethanol plus formic acid and ethanol plus hydrogen from glycerol at yields approaching their theoretical maximum.

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DNA microarrays are currently used to study the transcriptional response of many organisms to genetic and environmental perturbations. Although there is much room for improvement of this technology, its potential has been clearly demonstrated in the past 5 years. The general consensus is that the bottleneck is now located in the processing and analysis of transcriptome data and its use for purposes other than the quantification of changes in gene expression levels. In this article we discuss technological aspects of DNA microarrays, statistical and biological issues pertinent to the design of microarray experiments, and statistical tools for microarray data analysis. A review on applications of DNA microarrays in the study of bacterial systems is presented. Special attention is given to studies in the following areas: (1) bacterial response to environmental changes; (2) gene identification, genome organization, and transcriptional regulation; and (3) genetic and metabolic engineering. Soon, the use of DNA microarray technologies in conjunction with other genome/system-wide analyses (e.g., proteomics, metabolomics, fluxomics, phenomics, etc.) will provide a better assessment of genotype-phenotype relationships in bacteria, which serve as a basis for understanding similar processes in more complex organisms.