Kyriacos Zygourakis

Understanding and harnessing the microaerobic metabolism of glycerol in Escherichia coli

Given its availability, low prices, and high degree of reduction, glycerol has become an ideal feedstock for the production of reduced compounds. The anaerobic fermentation of glycerol by Escherichia coli could be an excellent platform for this purpose but it requires expensive nutrients such as tryptone and yeast extract. In this work, microaerobic conditions were used as a means of eliminating the need for rich nutrients. Availability of low amounts of oxygen enabled redox balance while preserving the ability to synthesize reduced products. A fermentation balance analysis showed ∼95% recovery of carbon and reducing equivalents. The pathways involved in glycerol dissimilation were identified using different genetic and biochemical approaches. Respiratory (GlpK‐GlpD/GlpABC) and fermentative (GldA‐DhaKLM) routes mediated the conversion of glycerol to glycolytic intermediates. Although pyruvate formate‐lyase (PFL) and pyruvate dehydrogenase contributed to the synthesis of acetyl‐CoA from pyruvate, most of the carbon flux proceeded through PFL. The pathways mediating the synthesis of acetate and ethanol were required for the efficient utilization of glycerol. The microaerobic metabolism of glycerol was harnessed by engineering strains for the co‐production of ethanol and hydrogen (EH05 [pZSKLMgldA]), and ethanol and formate (EF06 [pZSKLMgldA]). High ethanol yields were achieved by genetic manipulations that reduced the synthesis of by‐products succinate, acetate, and lactate. Co‐production of hydrogen required the use of acidic pH while formate co‐production was facilitated by inactivation of the enzyme formate‐hydrogen lyase. High rates of product synthesis were realized by overexpressing glycerol dehydrogenase (GldA) and dihydroxyacetone kinase (DhaKLM). Engineered strains efficiently produced ethanol and hydrogen and ethanol and formate from glycerol in a minimal medium without rich supplements. Biotechnol. Bioeng. 2009;103: 148–161.

Attachment, proliferation, and migration of marrow stromal osteoblasts cultured on biomimetic hydrogels modified with an osteopontin-derived peptide.

We prepared oligo(poly(ethylene glycol) fumarate) (OPF) hydrogels modified with a rat osteopontin-derived peptide (ODP), Asp-Val-Asp-Val-Pro-Asp-Gly-Arg-Gly-Asp-Ser-Leu-Ala-Try-Gly (DVDVPDGRGDSLAYG), as well as Gly-Arg-Gly-Asp-Ser (GRGDS) and investigated the modulation of marrow stromal osteoblast function on the peptide-modified hydrogels. Osteoblast attachment was competitively inhibited by a soluble peptide suggesting that the interaction of osteoblasts with the hydrogel was ligand specific. The proliferation index of osteoblasts relative to the initial seeding density was similar on the hydrogels modified with ODP (1.18+/-0.13) and GRGDS (1.27+/-0.12). However, fibroblasts proliferated faster on GRGDS-modified hydrogels than on ODP-modified hydrogels as evidenced by the proliferation indices of 4.89+/-0.03 and 2.42+/-0.16, respectively. A megacolony migration assay conducted for 3 days with a seeding density of 53,000 cells/cm(2) showed that osteoblasts migrated to a longer distance on ODP-modified hydrogels (0.23+/-0.06 mm/day) than on hydrogels modified with GRGDS (0.15+/-0.02 mm/day). In addition, osteoblasts migrated faster than fibroblasts seeded at the same density on ODP-modified hydrogels (0.15+/-0.11 mm/day). The migration of osteoblasts on the peptide-modified hydrogels was dependent on the peptide concentration of the hydrogels resulting in an increased migration distance with increasing the peptide concentration for the concentrations tested. These results show that OPF-based biomimetic hydrogels hold promise for modulating cell proliferation and migration for specific applications by altering the specific ligand and its concentration in the hydrogels.

Endothelial cell migration on surfaces modified with immobilized adhesive peptides

Endothelial cell (EC) migration has been studied on aminophase surfaces with covalently bound RGDS and YIGSRG cell adhesion peptides. The fluorescent marker dansyl chloride was used to quantify the spatial distribution of the peptides on the modified surfaces. Peptides appeared to be distributed in uniformly dispersed large clusters separated by areas of lower peptide concentrations. We employed digital time-lapse video microscopy and image analysis to monitor EC migration on the modified surfaces and to reconstruct the cell trajectories. The persistent random walk model was then applied to analyze the cell displacement data and compute the mean root square speed, the persistence time, and the random motility coefficient of EC. We also calculated the time-averaged speed of cell locomotion. No differences in the speed of cell locomotion on the various substrates were noted. Immobilization of the cell adhesion peptides (RGDS and YIGSRG), however, significantly increased the persistence of cell movement and, thus, the random motility coefficient. These results suggest that immobilization of cell adhesion peptides on the surface of implantable biomaterials may lead to enhanced endothelization rates.

Transient operation of monolith catalytic converters: a two-dimensional reactor model and the effects of radially nonuniform flow distributions

Abstract The transient operation of monolithic catalytic converters was stimulated using a two-dimensional equivalent continuum model that allowed us to systematically evaluate the effects of nonuniform flow disributions, of ambient heat losses, and of radially varying catalytic activity profiles on reactor performance. Non-uniform flow distributions characterized by high fluid velocities in an inner core of the reactor may substantially degrade its light-off performance, but such adverse effects can be alleviated by using “flow-tailoring” devices. The simulation results show that a step decrease in the feedstream temperature can lead to “wrong-way” behavior that here takes the form of large and highly localized overtemperature excursions. Ambient heat losses may substantially decrease the steady-state conversions obtained in a converter, although they only slightly retard the light-off process. Finally, the effects of radially nonuniform catalytic activity profiles are investigated. The presented results demonstrate that flow nonuniformities must be considered in comprehensive converter models together with other important design parameters that include the thermal mass of the reactor, exhaust gas temperature and stoichiometry, catalyst loading and gas—solid contact area.

Computer-aided design of bioerodible devices with optimal release characteristics: a cellular automata approach

The development of a computational tool to design bioerodible devices with optimal release characteristics is presented. This computational tool uses cellular automata and parallel iterations to model and simulate the release of bioactive agents (drugs) from bioerodible matrices. The simulations can accurately model surface erosion processes in multicomponent systems of arbitrary geometry and with different dissolution rates for each component. Simulation results are analysed to show how the overall release rates are affected by the intrinsic dissolution rates, drug loading, porosity and the dispersion of the drug in the bioerodible matrix. A strongly non-linear dependence of release rates on drug loading and the intrinsic dissolution rates of the solid components is obtained and the effects of phase dispersion on the variability of release rates are elucidated. Finally, guidelines are presented for screening of alternatives to minimize the development effort and experimentation required for designing devices with desired release characteristics.

Biochar and Microbial Signaling: Production Conditions Determine Effects on Microbial Communication

Charcoal has a long soil residence time, which has resulted in its production and use as a carbon sequestration technique (biochar). A range of biological effects can be triggered by soil biochar that can positively and negatively influence carbon storage, such as changing the decomposition rate of organic matter and altering plant biomass production. Sorption of cellular signals has been hypothesized to underlie some of these effects, but it remains unknown whether the binding of biochemical signals occurs, and if so, on time scales relevant to microbial growth and communication. We examined biochar sorption of N-3-oxo-dodecanoyl-L-homoserine lactone, an acyl-homoserine lactone (AHL) intercellular signaling molecule used by many gram-negative soil microbes to regulate gene expression. We show that wood biochars disrupt communication within a growing multicellular system that is made up of sender cells that synthesize AHL and receiver cells that express green fluorescent protein in response to an AHL signal. However, biochar inhibition of AHL-mediated cell-cell communication varied, with the biochar prepared at 700 °C (surface area of 301 m(2)/g) inhibiting cellular communication 10-fold more than an equivalent mass of biochar prepared at 300 °C (surface area of 3 m(2)/g). These findings provide the first direct evidence that biochars elicit a range of effects on gene expression dependent on intercellular signaling, implicating the method of biochar preparation as a parameter that could be tuned to regulate microbial-dependent soil processes, like nitrogen fixation and pest attack of root crops.

A cellular automaton model for the proliferation of migrating contact-inhibited cells

A cellular automaton is used to develop a model describing the proliferation dynamics of populations of migrating, contact-inhibited cells. Simulations are carried out on two-dimensional networks of computational sites that are finite-state automata. The discrete model incorporates all the essential features of the cell locomotion and division processes, including the complicated dynamic phenomena occurring when cells collide. In addition, model parameters can be evaluated by using data from long-term tracking and analysis of cell locomotion. Simulation results are analyzed to determine how the competing processes of contact inhibition and cell migration affect the proliferation rates. The relation between cell density and contact inhibition is probed by following the temporal evolution of the population-average speed of locomotion. Our results show that the seeding cell density, the population-average speed of locomotion, and the spatial distribution of the seed cells are crucial parameters in determining the temporal evolution of cell proliferation rates. The model successfully predicts the effect of cell motility on the growth of isolated megacolonies of keratinocytes, and simulation results agree very well with experimental data. Model predictions also agree well with experimentally measured proliferation rates of bovine pulmonary artery endothelial cells (BPAE) cultured in the presence of a growth factor (bFGF) that up-regulates cell motility.

Quantitative analysis of the fermentative metabolism of glycerol in Escherichia coli

Availability, low price, and high degree of reduction have made glycerol a highly attractive and exploited carbon source for the production of fuels and reduced chemicals. Here we report the quantitative analysis of the fermentative metabolism of glycerol in Escherichia coli through the use of kinetic modeling and metabolic control analysis (MCA) to gain a better understanding of glycerol fermentation and identify key targets for genetic manipulation that could enhance product synthesis. The kinetics of glycerol fermentation in a batch culture was simulated using a dynamic model consisting of mass balances for glycerol, ethanol, biomass, and 11 intracellular metabolites, along with the corresponding kinetic expressions for the metabolism of each species. The model was then used to calculate metabolic control coefficients and elucidate the control structure of the pathways involved in glycerol utilization and ethanol synthesis. The calculated flux control coefficients indicate that the glycolytic flux during glycerol fermentation is almost exclusively controlled by the enzymes glycerol dehydrogenase (encoded by gldA) and dihydroxyacetone kinase (DHAK) (encoded by dhaKLM). In agreement with the MCA findings, overexpression of gldA and dhaKLM led to significant increase in glycerol utilization and ethanol synthesis fluxes. Moreover, overexpression of other enzymes involved in the pathways that mediate glycerol utilization and its conversion to ethanol had no significant impact on glycerol utilization and ethanol synthesis, further validating the MCA predictions. These findings were then applied as a means of increasing the production of ethanol: overexpression of glycerol dehyrdogenase and DHAK enabled the production of 20 g/L ethanol from crude glycerol, a by‐product of biodiesel production, indicating the potential for industrial scale conversion of waste glycerol to ethanol under anaerobic conditions. Biotechnol. Bioeng. 2012;109: 187–198.

Development and temporal evolution of erosion fronts in bioerodible controlled release devices

Cellular automata and discrete iterations are used to model and simulate the release of bioactive agents (drugs) from bioerodible pellets used as controlled release devices. The presented discrete simulation approach is a computationally efficient tool for designing delivery systems with optimal release properties, since it can accurately treat the transient erosion processes in multicomponent release systems of arbitrary geometry and with different dissolution rates for each component. Results from two-dimensional simulations are presented to show how the intrinsic dissolution rates, drug loadings, matrix porosities etc. affect the overall release rates and to establish the relationship between the ratio of dissolution rates and the fractal dimension of the erosion front.

Migration of lymphocytes on fibronectin-coated surfaces: temporal evolution of migratory parameters.

Lymphocytes typically interact with implanted biomaterials through adsorbed exogenous proteins. To provide a more complete characterization of these interactions, analysis of lymphocyte migration on adsorbed extracellular matrix proteins must accompany the commonly performed adhesion studies. We report here a comparison of the migratory and adhesion behavior of Jurkat cells (a T lymphoblastoid cell line) on tissue culture treated and untreated polystyrene surfaces coated with various concentrations of fibronectin. The average speed of cell locomotion showed a biphasic response to substrate adhesiveness for cells migrating on untreated polystyrene and a monotonic decrease for cells migrating on tissue culture-treated polystyrene. A modified approach to the persistent random walk model was implemented to determine the time dependence of cell migration parameters. The random motility coefficient showed significant increases with time when cells migrated on tissue culture-treated polystyrene surfaces, while it remained relatively constant for experiments with untreated polystyrene plates. Finally, a cell migration computer model was developed to verify our modified persistent random walk analysis. Simulation results suggest that our experimental data were consistent with temporally increasing random motility coefficients.