Microbially produced fatty acids are potential precursors to high-energy-density biofuels, including alkanes and alkyl ethyl esters, by either catalytic conversion of free fatty acids (FFAs) or enzymatic conversion of acyl-acyl carrier protein or acyl-coenzyme A intermediates. Metabolic engineering efforts aimed at overproducing FFAs in Escherichia coli have achieved less than 30% of the maximum theoretical yield on the supplied carbon source. In this work, the viability, morphology, transcript levels, and protein levels of a strain of E. coli that overproduces medium-chain-length FFAs was compared to an engineered control strain. By early stationary phase, an 85% reduction in viable cell counts and exacerbated loss of inner membrane integrity were observed in the FFA-overproducing strain. These effects were enhanced in strains endogenously producing FFAs compared to strains exposed to exogenously fed FFAs. Under two sets of cultivation conditions, longchain unsaturated fatty acid content greatly increased, and the expression of genes and proteins required for unsaturated fatty acid biosynthesis were significantly decreased. Membrane stresses were further implicated by increased expression of genes and proteins of the phage shock response, the MarA/Rob/SoxS regulon, and the nuo and cyo operons of aerobic respiration. Gene deletion studies confirmed the importance of the phage shock proteins and Rob for maintaining cell viability; however, little to no change in FFA titer was observed after 24 h of cultivation. The results of this study serve as a baseline for future targeted attempts to improve FFA yields and titers in E. coli.Microbially derived free fatty acids (FFAs) are attractive intermediates for producing a wide range of high-energy-density biofuels from sustainable carbon sources, such as biomass (34). FFAs can be extracted from culture medium and catalytically converted to esters or alkanes (48, 55). Alternatively, enzymatic pathways exist for intracellular conversion to esters (42, 81), olefins (10, 59, 75), alkanes (78), or fatty aldehydes and fatty alcohols (22,81,82). These pathways can either be exploited in their native host or heterologously expressed in a genetically pliable microorganism (3). The physical and chemical properties of the resulting products are dependent on chain length and hydrophobicity; however, medium-chainlength (8-to 14-carbon) methyl esters, olefins, and alkanes exhibit many properties analogous to those of diesel and jet fuel and are therefore potential drop-in replacements (44,61).Several studies have demonstrated FFA overproduction in Escherichia coli (19,48,52,81,83). In each, the key strain modifications included overexpression of one or more cytosolic acyl-acyl carrier protein (ACP) thioesterases and deletion of fadD, or both fadD and fadE, which encode an acyl-coenzyme A (CoA) synthetase and acyl-CoA dehydrogenase, respectively. Overexpression of an acyl-ACP thioesterase depletes the level of acyl-ACP intermediates, which inhibit via feedback enzymes of fatty acid bi...

Starting from stationary bifurcations in Couette-Dean flow, we compute nontrivial stationary solutions in inertialess viscoelastic circular Couette flow. These solutions are strongly localized vortex pairs, exist at arbitrarily large wavelengths, and show hysteresis in the Weissenberg number, similar to experimentally observed "diwhirl" patterns. Based on the computed velocity and stress fields, we elucidate a heuristic, fully nonlinear mechanism for these flows. We propose that these localized, fully nonlinear structures comprise fundamental building blocks for complex spatiotemporal dynamics in the flow of elastic liquids. Flow instabilities and nonlinear dynamics have long been recognized to occur in flows of viscoelastic polymer melts and solutions [1][2][3]. An important breakthrough, which has led to increasing recent attention to these phenomena, was made by Larson, Shaqfeh and Muller [4], who discovered that circular Couette flow of a viscoelastic liquid undergoes an instability loosely analogous to the classical Taylor-Couette instability of Newtonian liquids, but driven solely by elasticity -the instability is present at zero Taylor (Reynolds) number. They also showed that a linear stability analysis of a simple fluid model predicts instability and elucidated the basic elasticity-driven mechanism of the instability. In particular, although many researchers had studied the effects of viscoelasticity on the Newtonian (inertial) Taylor-Couette instability, both experimentally [5][6][7][8][9], and theoretically [10,11,[5][6][7]12], these workers were the first to demonstrate an inertialess, purely elastic mechanism for instability in a viscometric flow. More recent observations have revealed a wealth of interesting dynamics in this flow as well as other simple flows [3,[13][14][15]; one set of experimental observations of particular interest was made by Groisman and Steinberg [13], who found long-wavelength, stationary axisymmetric vortex pair structures in inertialess viscoelastic flow in the circular Couette geometry. There are two interesting aspects to these observations: (1) isothermal linear stability analysis in this geometry never predicts bifurcation of stationary states (in contrast to the classical Taylor-Couette case) and (2) the observations suggest that these vortex pairs, which Groisman and Steinberg dubbed "diwhirls", can exist in isolation -there does not seem to be a selected axial wavelength for this pattern. These considerations motivate the present computational study, which addresses the following questions: (1) Do isolated branches of stationary solutions exist in a simple model of a viscoelastic fluid in the circular Couette geometry? (2) If so, what are the spatial structures of these? Are they localized? (3) A nonlinear self-sustaining mechanism must be present for such patterns to exist. Can the computations help us elucidate it?We address these questions by fully nonlinear computations of the branching behavior of an inertialess isothermal FENE dumbbell fluid in the circ...

Starting from stationary bifurcations in Couette–Dean flow, we compute stationary nontrivial solutions in the circular Couette geometry for an inertialess finitely extensible nonlinear elastic (FENE-P) dumbbell fluid. These solutions are isolated from the Couette flow branch arising at finite amplitude in saddle–node bifurcations as the Weissenberg number increases. Spatially, they are strongly localized axisymmetric vortex pairs embedded in an arbitrarily long ‘far field’ of pure Couette flow, and are thus qualitatively, and to some extent quantitatively, similar to the ‘diwhirl’ (Groisman & Steinberg 1997) and ‘flame’ patterns (Baumert & Muller 1999) observed experimentally. For computationally accessible parameter values, these solutions appear only above the linear instability limit of the Couette base flow, in contrast to the experimental observations. Correspondingly, they are themselves linearly unstable. Nevertheless, extrapolation of the trend in the bifurcation points with increasing polymer extensibility suggests that for sufficiently high extensibility the diwhirls will come into existence before the linear instability, as seen experimentally.Based on the computed stress and velocity fields, we propose a fully nonlinear self-sustaining mechanism for these flows. The mechanism is related to that for viscoelastic Dean flow vortices and arises from a finite-amplitude perturbation giving rise to a locally unstable profile of the azimuthal normal stress near the outer cylinder at the symmetry plane of the vortex pair. The unstable stress profile, in combination with a ‘tubeless siphon’ effect, nonlinearly sustains the patterns. We propose that these solitary, strongly nonlinear structures comprise fundamental building blocks for complex spatiotemporal dynamics in the flow of elastic liquids.

Experiments and theory show that hydrodynamic instabilities can arise during flow of viscoelastic liquids in curved geometries. A recent study has found that a relatively weak steady transverse flow can delay the onset of instability in the circular Couette geometry until the azimuthal Weissenberg number Weθ is significantly higher than without axial flow. In this work we investigate the effect of superposition of a time-periodic axial Couette flow on the viscoelastic circular Couette and Dean flow instabilities. The analysis, carried out for the upper-convected Maxwell and Oldroyd-B fluids, generally shows increased stability compared to when there is no axial flow. However, we also find that the system shows instability – synchronous resonance – for some values of the axial Weissenberg number, Wez and forcing frequency ω. In particular, instability can be induced not only when ω is of the order of the inverse relaxation time of the fluid but also when it is much smaller. Scaling arguments and numerical results indicate that the high-ω, low-Wez regime is essentially equivalent to Wez=0 in the steady case, implying no stabilization. At high values of ω and Wez, scaling analysis shows that the flow will always be stable. Numerical results are in agreement with these conclusions. Consistent with previous results on parametrically forced systems, we find that the zero-frequency limit is singular. In this limit, the disturbances display quiescent intervals punctuated by periods of large transient growth and subsequent decay.This study also presents linear and nonlinear stability results for the addition of steady axial Couette and Poiseuille flows to viscoelastic instabilities in azimuthal Dean flows. It is shown that, for high Wez, the qualitative effect of adding a steady axial flow is similar to that in the circular Couette geometry, with a linear relationship between the critical Weθ and Wez. For low Wez, we find that the flow is stabilized, unlike in the circular Couette flow where the critical value of Weθ decreases at low Wez. Further, weakly nonlinear analysis shows that the criticality of the bifurcation depends on the value of Wez and the solvent viscosity, S. Finally, we also show the presence of a codimension-2 Takens–Bogdanov bifurcation point in the linear stability curve of Dean flow. This point represents a transition from one mechanism of instability to another.

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