Current Flow in a Cylindrical Nanopore with an Object-Implications for Virus Sensing.

Interest is growing in nanopores as real-time, low-cost, label-free virus size sensors. To optimize their performance, we evaluate how external electric field and ion concentrations and pore wall charges influence currents and object (disk) radius-current relationship using simulations. The physics was described using the Poisson-Nernst-Planck and Navier-Stokes equations. In a charged cylindrical nanopore with a charged disk, elevated external electric field produces higher (and polarity independent) ion concentrations and greater ion current (largely migratory). Elevated external ion concentrations also lead to higher concentrations (mainly away from the pore wall), greater axial electric field especially in the disk-pore wall space, and finally larger current. At low concentrations, current is disk radius independent. The current rises as concentrations increase. Interestingly, the rise is greater for larger disks (except when the pore is blocked mechanically). Smaller cross-sectional area for current flow or volume exclusion of electrolyte by object thus cannot be universally accepted as explanations of current blockage. Ion current rises when pore wall charge density increases, but its direction is independent of charge sign. Current-disk radius relationship is also independent of pore wall charge sign. If the pore wall and disk charges have the same sign, larger current with bigger disk is due to higher counter-ion accumulation in the object-pore wall space. However, if their signs are opposite, it is largely due to elevated axial electric field in the object-pore wall space. Finally in uncharged nanopores, current diminishes when disk radius increases making them better sensors of virus size.

Axial forces at disk surfaces in a cylindrical nanopore.

Understanding the physics of object translocation in nanopores is critical for using nanopores as sensors of molecular properties and as object size and shape sensors. Based on Poisson-Nernst-Planck and Navier-Stokes simulations we dissect three axial pressures and forces at disk edges (upper, lower and rim) - Coulomb, dielectric and fluidic. Axial Coulomb and dielectric rim forces are small and cancel each other. Upper and lower axial forces are largely controlled by the external axial electric field and interestingly by the pore wall charges that determine the amplitude and direction of axial combined force. Axial total Coulomb force (sum of its upper and lower edge components) makes the greatest contribution, but the axial total dielectric force (calculated using Maxwell stress tensor), which opposes it is surprisingly large. External ion concentration alters Coulomb and axial dielectric forces but influences only their amplitude. Axial total fluidic force is near zero (its upper and lower disk edge components are significant but cancel each other) regardless of external electric field, but pore wall charges and external fluidic pressure can alter it. Modest changes of external electric field or concentration produce axial forces comparable to those produced by large external fluidic pressures. Axial forces depend little on disk's axial position. Finally, mean axial pressures (calculated to compare forces acting on disks of different radius) are greater for larger disks.

Pilot Scale Study: First Demonstration of Hydrophobic Membranes for the Removal of Ammonia Molecules from Rendering Condensate Wastewater.

Hydrophobic membrane contactors represent a promising solution to the problem of recycling ammoniacal nitrogen (N-NH4) molecules from waste, water or wastewater resources. The process has been shown to work best with wastewater streams that present high N-NH4 concentrations, low buffering capacities and low total suspended solids. The removal of N-NH4 from rendering condensate, produced during heat treatment of waste animal tissue, was assessed in this research using a hydrophobic membrane contactor. This study investigates how the molecular composition of rendering condensate wastewater undergo changes in its chemistry in order to achieve suitability to be treated using hydrophobic membranes and form a suitable product. The main objective was to test the ammonia stripping technology using two types of hydrophobic membrane materials, polypropylene (PP) and polytetrafluoroethylene (PTFE) at pilot scale and carry out: (i) Process modification for NH3 molecule removal and (ii) product characterization from the process. The results demonstrate that PP membranes are not compatible with the condensate waste as it caused wetting. The PTFE membranes showed potential and had a longer lifetime than the PP membranes and removed up to 64% of NH3 molecules from the condensate waste. The product formed contained a 30% concentrated ammonium sulphate salt which has a potential application as a fertilizer. This is the first demonstration of hydrophobic membrane contactors for treatment of condensate wastewater.

Effect of ion concentration, solution and membrane permittivity on electric energy storage and capacitance.

Bio-membranes as capacitors store electric energy, but their permittivity is low whereas the permittivity of surrounding solution is high. To evaluate the effective capacitance of the membrane/solution system and determine the electric energy stored within the membrane and in the solution, we estimated their electric variables using Poisson-Nernst-Planck simulations. We calculated membrane and solution capacitances from stored electric energy. The effective capacitance was calculated by fitting a six-capacitance model to charges (fixed and ion) and associated potentials, because it cannot be considered as a result of membrane and solution capacitance in series. The electric energy stored within the membrane (typically much smaller than that in the solution), depends on the membrane permittivity, but also on the external electric field, surface charge density, water permittivity and ion concentration. The effect on capacitances is more specific. Solution capacitance rises with greater solution permittivity or ion concentration, but the membrane capacitance (much smaller than solution capacitance) is only influenced by its permittivity. Interestingly, the effective capacitance is independent of membrane or solution permittivity, but rises as the ion concentration increases and surface charge becomes positive. Experimental estimates of membrane capacitance are thus not necessarily a reliable index of its surface area.

Predicting the accumulation of storage compounds by Rhodococcus jostii RHA1 in the feast-famine growth cycles using genome-scale flux balance analysis.

Feast-famine cycles in biological wastewater resource recovery systems select for bacterial species that accumulate intracellular storage compounds such as poly-ß-hydroxybutyrate (PHB), glycogen, and triacylglycerols (TAG). These species survive better the famine phase and resume rapid substrate uptake at the beginning of the feast phase faster than microorganisms unable to accumulate storage. However, ecophysiological conditions favouring the accumulation of either storage compounds remain to be clarified, and predictive capabilities need to be developed to eventually rationally design reactors producing these compounds. Using a genome-scale metabolic modelling approach, the storage metabolism of Rhodococcus jostii RHA1 was investigated for steady-state feast-famine cycles on glucose and acetate as the sole carbon sources. R. jostii RHA1 is capable of accumulating the three storage compounds (PHB, TAG, and glycogen) simultaneously. According to the experimental observations, when glucose was the substrate, feast phase chemical oxygen demand (COD) accumulation was similar for the three storage compounds; when acetate was the substrate, however, PHB accumulation was 3 times higher than TAG accumulation and essentially no glycogen was accumulated. These results were simulated using the genome-scale metabolic model of R. jostii RHA1 (iMT1174) by means of flux balance analysis (FBA) to determine the objective functions capable of predicting these behaviours. Maximization of the growth rate was set as the main objective function, while minimization of total reaction fluxes and minimization of metabolic adjustment (environmental MOMA) were considered as the sub-objective functions. The environmental MOMA sub-objective performed better than the minimization of total reaction fluxes sub-objective function at predicting the mixture of storage compounds accumulated. Additional experiments with 13C-labelled bicarbonate (HCO3-) found that the fluxes through the central metabolism reactions during the feast phases were similar to the ones during the famine phases on acetate due to similarity in the carbon sources in the feast and famine phases (i.e., acetate and poly-ß-hydroxybutyrate, respectively); this suggests that the environmental MOMA sub-objective function could be used to analyze successive environmental conditions such as the feast and famine cycles while the metabolically similar carbon sources are taken up by microorganisms.

Genome-scale metabolic model of Rhodococcus jostii RHA1 (iMT1174) to study the accumulation of storage compounds during nitrogen-limited condition.

BACKGROUND: Rhodococcus jostii RHA1 growing on different substrates is capable of accumulating simultaneously three types of carbon storage compounds: glycogen, polyhydroxyalkanoates (PHA), and triacylglycerols (TAG). Under nitrogen-limited (N-limited) condition, the level of storage increases as is commonly observed for other bacteria. The proportion of each storage compound changes with substrate, but it remains unclear what modelling approach should be adopted to predict the relative composition of the mixture of the storage compounds. We analyzed the growth of R. jostii RHA1 under N-limited conditions using a genome-scale metabolic modelling approach to determine which global metabolic objective function could be used for the prediction. RESULTS: The R. jostii RHA1 model (iMT1174) produced during this study contains 1,243 balanced metabolites, 1,935 unique reactions, and 1,174 open reading frames (ORFs). Seven objective functions used with flux balance analysis (FBA) were compared for their capacity to predict the mixture of storage compounds accumulated after the sudden onset of N-limitation. Predictive abilities were determined using a Bayesian approach. Experimental data on storage accumulation mixture (glycogen, polyhydroxyalkanoates, and triacylglycerols) were obtained for batch cultures grown on glucose or acetate. The best FBA simulation results were obtained using a novel objective function for the N-limited condition which combined the maximization of the storage fluxes and the minimization of metabolic adjustments (MOMA) with the preceding non-limited conditions (max storage + environmental MOMA). The FBA solutions for the non-limited growth conditions were simply constrained by the objective function of growth rate maximization. Measurement of central metabolic fluxes by (13)C-labelling experiments of amino acids further supported the application of the environmental MOMA principle in the context of changing environment. Finally, it was found that the quantitative predictions of the storage mixture during N-limited storage accumulation were fairly sensitive to the biomass composition, as expected. CONCLUSIONS: The genome-scale metabolic model analysis of R. jostii RHA1 cultures suggested that the intracellular reaction flux profile immediately after the onset of N-limited condition are impacted by the values of the same fluxes during the period of non-limited growth. PHA turned out to be the main storage pool of the mixture in R. jostii RHA1.

Spatial profiles of potential, ion concentration and flux in short unipolar and bipolar nanopores.

During release of vesicular content the resistance of the fusion pore sometimes changes rapidly and repeatedly. However, it is not clear why the pore 'flickers'. Engineered nanopores often rectify, but how different factors influence the rectification requires clarification. To better understand the ionic 'causes' of pore conductivity and its changes we simulated ion transport through a short nanopore using Poisson-Nernst-Planck equations, coupling it to the transport of water using Navier-Stokes equations. We extracted the potential, concentration, and ion flux profiles. In uniformly charged nanopores the voltage bias determines which counter-ion flux dominates, and it is carried by the counter-ions of the highest concentration. In unipolar nanopores this simple rule breaks down. The dominant counter-ion in the charged half is from the adjacent compartment, but the bias determines what counter-ion flux is dominant--the same ion (regular bias), or a different and smaller (reverse bias), and this difference determines the level of rectification. In bipolar nanopores the dominant counter-ions in each half are from the adjacent compartments, and the total ion concentration dips in the middle near the wall. With regular bias the total ion concentration peaks in the pore center; the ions that carry the current through the nanopore start as counter-ions and their fluxes are large. With reverse bias the total concentration dips near the wall and in the center, both dominant ion fluxes through the nanopore start as co-ions and are very small, whereas those starting as counter-ions do not go through.

Genome-scale metabolic modeling to provide insight into the production of storage compounds during feast-famine cycles of activated sludge.

Studying storage metabolism during feast-famine cycles of activated sludge treatment systems provides profound insight in terms of both operational issues (e.g., foaming and bulking) and process optimization for the production of value added by-products (e.g., bioplastics). We examined the storage metabolism (including poly-ß-hydroxybutyrate [PHB], glycogen, and triacylglycerols [TAGs]) during feast-famine cycles using two genome-scale metabolic models: Rhodococcus jostii RHA1 (iMT1174) and Escherichia coli K-12 (iAF1260) for growth on glucose, acetate, and succinate. The goal was to develop the proper objective function (OF) for the prediction of the main storage compound produced in activated sludge for given feast-famine cycle conditions. For the flux balance analysis, combinations of three OFs were tested. For all of them, the main OF was to maximize growth rates. Two additional sub-OFs were used: (1) minimization of biochemical fluxes, and (2) minimization of metabolic adjustments (MoMA) between the feast and famine periods. All (sub-)OFs predicted identical substrate-storage associations for the feast-famine growth of the above-mentioned metabolic models on a given substrate when glucose and acetate were set as sole carbon sources (i.e., glucose-glycogen and acetate-PHB), in agreement with experimental observations. However, in the case of succinate as substrate, the predictions depended on the network structure of the metabolic models such that the E. coli model predicted glycogen accumulation and the R. jostii model predicted PHB accumulation. While the accumulation of both PHB and glycogen was observed experimentally, PHB showed higher dynamics during an activated sludge feast-famine growth cycle with succinate as substrate. These results suggest that new modeling insights between metabolic predictions and population ecology will be necessary to properly predict metabolisms likely to emerge within the niches of activated sludge communities. Nonetheless, we believe that the development of this approach will help guide the optimization of the production of storage compounds as valuable by-products of wastewater treatment.