Fermentative utilization of glycerol by Escherichia coli and its implications for the production of fuels and chemicals

Abstract

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-13C-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-13C-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 (approximately 95%) and redox (approximately 96%) balances. On the other hand, cultivation conditions that prevent H2 accumulation were shown to be an environmental determinant of glycerol fermentation. The negative effect of H2 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.

FIG. 1.

Respiratory metabolism of glycerol in E. coli. Glycerol dissimilation in the presence of electron acceptors is mediated by an ATP-dependent glycerol kinase (GK, coded for by glpK) and two respiratory G3PDHs (aerobic and anaerobic enzymes, encoded by glpD and glpABC, respectively). Abbreviations: DHAP, dihydroxyacetone phosphate; GK, glycerol kinase; ae-G3PDH, aerobic G3PDH; H, reducing equivalents (H = NADH/NADPH/FADH₂); PYR, pyruvate.

FIG. 2.

Fermentation of glycerol by E. coli MG1655 in MM supplemented with 2 g/liter tryptone. (A) Cell density (▪ and □, for linear and log-linear plots, respectively) and concentrations of glycerol (▴), ethanol (•), succinic acid (♦), and formic plus acetic acids (×). Additional measurements of OD taken during the exponential growth phase are shown in the log-linear plot, along with their fitting to a straight-line model (least-squares method). (B) 1D ¹H NMR spectrum of the culture medium in a late fermentation sample. (Inset I) Two peaks at the same chemical shifts as those of methyl protons of 1,2-PDO (doublet at 1.15 ppm) are shown (*). (Inset II) Spectra for the time zero sample. (C) 1D ¹H NMR spectra of the fermentation broth from a 72-h culture grown on a mixture of 50% U-¹³C-labeled and 50% unlabeled glycerol. The percentage of area of each peak to that of total area (i.e., sum of all peaks) is shown.

FIG. 3.

NMR spectra of proteinogenic amino acids in cell biomass obtained from experiments with 50% U-¹³C-labeled (top panels) and unlabeled (bottom panels) glycerol. The identity of ¹³C satellites as peaks arising due to labeled carbon was confirmed by performing a ¹³C-decoupled experiment in which the ¹³C signals were suppressed (middle panels). Marked peaks (arrows) illustrate the incorporation of labeled carbon into threonine-γ (left panels), alanine-β (center panels), and glutamate-γ (right panels). Peaks in bottom panels (unlabeled glycerol) correspond to the natural abundance of ¹³C.

FIG. 4.

Effect of pH, carbon dioxide and hydrogen on glycerol fermentation. Fermented glycerol (line) and cell growth (bars) are shown. Bar color indicates pH 6.3 (gray) or 7.5 (clear). Values represent the means and error bars represent the standard deviations for samples taken once the cultures reached stationary phase. Experiments were conducted at 37°C and using MM supplemented with 10 g/liter glycerol and 2 g/liter tryptone. The composition of the gas atmosphere is as indicated. The same results were obtained by inclusion of hydrogen as either 20% balance argon or pure hydrogen.

FIG. 5.

Effect of gas atmosphere and hydrogen recycling on the internal redox state. The ΔfrdA mutant is devoid of the enzyme fumarate reductase, which converts fumarate to succinate. Calculated P values (t distribution) for the comparison of the NADH/NAD⁺ ratios are also shown.

FIG. 6.

Coproduction of ethanol (solid circles) and H₂ (gray bars) and ethanol (solid circles) and formic acid (clear bars) from glycerol in wild-type and engineered strains. Fermented glycerol (solid bars) and cell growth (solid triangles) are also shown. Reported values are averages of triplicate measurements, and error bars represent standard deviations. Argon was used as the headspace gas. HycB is a required component of the FHL system.

FIG. 7.

Conversion of glycerol and xylose to fermentation products. The degree of reduction per carbon for xylose and glycerol is shown in parentheses and was estimated as described elsewhere (19). Enzymes catalyzing relevant steps are shown (encoding genes in parentheses). The use of NADH or H₂ as an electron donor in the reduction of fumarate to succinate involves several proteins/enzymes, including fumarate reductase, NADH dehydrogenase, hydrogenases, and menaquinones (see Fig. 8 for details). Boxed metabolites are extracellular. Abbreviations: ACK, acetate kinase; ADH, alcohol/acetaldehyde dehydrogenase; LDH, lactate dehydrogenase; PFL, pyruvate-formate lyase; PTA, phosphate acetyltransferase; and PYK, pyruvate kinase.

FIG. 8.

Generation and recycling of hydrogen and their relationship to succinate production during the fermentative metabolism of glycerol. The use of hydrogen as an electron donor in the reduction of fumarate makes the conversion of glycerol to succinate a “redox-generating” pathway. See Fig. 7 for details about ATP use/generation in these pathways. Abbreviations: Hyd, hydrogenases I and II; ND, NADH dehydrogenase; and Q, quinone pool.

FIG. 9.

Anaerobic fermentation of glycerol as a platform for the production of fuels and reduced chemicals. Synthesis of these products from glycerol is a redox-balanced or redox-consuming process. The synthesis of ethanol or butanol along with either CO₂-H₂ or formate would generate ATP. Engineering the remaining pathways would involve their transformation to either ATP-generating (succinate and propionate) or ATP-neutral (1,2-PDO and isopropanolamine) pathways. The maximum theoretical yield in each case is higher than that possible from the use of common sugars such as glucose or xylose. The butanol pathway is not native in E. coli. Abbreviations: AcCoA, acetyl-CoA; DHAP, dihydroxyacetone phosphate; PEP, phosphoenolpyruvate; PYR, pyruvate; and 1,2-PDO, 1,2-propanediol.