Microfluidic Platform with Serpentine Geometry Providing Chaotic Mixing in Induction Time Experiments.

We present a droplet microfluidic platform mixing the contents of the droplet chaotically in microfluidic induction time measurements, a promising method for quantifying nucleation kinetics with minute amounts of solute. The nucleation kinetics of aqueous potassium chloride droplets dispersed in mineral oil without surfactants is quantified in the presence and absence of chaotic mixing. We demonstrate the ability of the proposed platform to dictate droplet size, to provide a homogeneous temperature distribution, and to chaotically mix the droplet contents.

Experimental design for evaluating WWTP data by linear mass balances.

A stepwise experimental design procedure to obtain reliable data from wastewater treatment plants (WWTPs) was developed. The proposed procedure aims at determining sets of additional measurements (besides available ones) that guarantee the identifiability of key process variables, which means that their value can be calculated from other, measured variables, based on available constraints in the form of linear mass balances. Among all solutions, i.

Extreme calorie restriction in yeast retentostats induces uniform non-quiescent growth arrest.

Non-dividing Saccharomyces cerevisiae cultures are highly relevant for fundamental and applied studies. However, cultivation conditions in which non-dividing cells retain substantial metabolic activity are lacking. Unlike stationary-phase (SP) batch cultures, the current experimental paradigm for non-dividing yeast cultures, cultivation under extreme calorie restriction (ECR) in retentostat enables non-dividing yeast cells to retain substantial metabolic activity and to prevent rapid cellular deterioration.

Growth inhibition of S. cerevisiae, B. subtilis, and E. coli by lignocellulosic and fermentation products.

This paper describes the effect of several inhibiting components on three potential hosts for the bio-based production of methyl propionate, namely, wild-type Escherichia coli and Bacillus subtilis, and evolved Saccharomyces cerevisiae IMS0351. The inhibition by the lignocellulose-derived products 5-hydroxymethyl-2-furaldehyde, vanillin, and syringaldehyde and the fermentation products 2-butanol, 2-butanone, methyl propionate, and ethyl acetate has been assessed for these strains in defined medium. Multiple screenings were performed using small-scale cultures in both shake flasks and microtiter plates.

In vivo kinetic analysis of the penicillin biosynthesis pathway using PAA stimulus response experiments.

In this study we combined experimentation with mathematical modeling to unravel the in vivo kinetic properties of the enzymes and transporters of the penicillin biosynthesis pathway in a high yielding Penicillium chrysogenum strain. The experiment consisted of a step response experiment with the side chain precursor phenyl acetic acid (PAA) in a glucose-limited chemostat. The metabolite data showed that in the absence of PAA all penicillin pathway enzymes were expressed, leading to the production of a significant amount of 6-aminopenicillanic acid (6APA) as end product.

Mathematical model of adherent Vero cell growth and poliovirus production in animal component free medium.

Sabin-IPV (or sIPV, inactivated polio vaccine based on attenuated Sabin strains) is anticipated to replace the oral polio vaccine for the endgame in polio eradication. Optimization of sIPV production will lead to a better economically feasible vaccine. To assist process optimization, we studied Sabin type 1 poliovirus (PV) infection kinetics on Vero cells in controlled bioreactor vessels.

Thermodynamic description of peptide adsorption on mixed-mode resins.

In this work the adsorption of tri-peptides on a mixed-mode resin was studied using isocratic pulse response experiments. Various salt concentration, temperature and pH combinations were used to measure retention times of several tri-peptides. The experiments were evaluated according to an extension of the stoichiometric displacement model and the steric mass action model of protein-ligand binding.

Parameter identification of in vivo kinetic models: limitations and challenges.

Systems metabolic engineering of metabolic networks by genetic techniques requires kinetic equations for each enzyme present. In vitro studies of singular enzymes have limitations for predicting in vivo behavior, and in vivo experiments are constrained to retain viable cells. The estimation of kinetic parameters in vivo is a challenge due to the complexity of the internal cell environment.

Efficient calculation of compound similarity based on maximum common subgraphs and its application to prediction of gene transcript levels.

Properties of a chemical entity, both physical and biological, are related to its structure. Since compound similarity can be used to infer properties of novel compounds, in chemoinformatics much attention has been paid to ways of calculating structural similarity. A useful metric to capture the structural similarity between compounds is the relative size of the Maximum Common Subgraph (MCS).

Microbial diversity differences within aerobic granular sludge and activated sludge flocs.

In this study, we investigated during 400 days the microbial community variations as observed from 16S DNA gene DGGE banding patterns from an aerobic granular sludge pilot plant as well as the from a full-scale activated sludge treatment plant in Epe, the Netherlands. Both plants obtained the same wastewater and had the same relative hydraulic variations and run stable over time. For the total bacterial population, a similarity analysis was conducted showing that the community composition of both sludge types was very dissimilar.

Use of sequential-batch fermentations to characterize the impact of mild hypothermic temperatures on the anaerobic stoichiometry and kinetics of Saccharomyces cerevisiae.

This work presents a characterization of the stoichiometry and kinetics of anaerobic batch growth of Saccharomyces cerevisiae at cultivation temperatures between 12 and 30°C. To minimize the influence of the inoculum condition and ensure full adaptation to the cultivation temperature, the experiments were carried out in sequencing batch reactors. It was observed that the growth rate obtained in the first batch performed after each temperature shift was 10-30% different compared with the subsequent batches at the same temperature, which were much more reproducible.

Model-based rational strategy for chromatographic resin selection.

A model-based rational strategy for the selection of chromatographic resins is presented. The main question being addressed is that of selecting the most optimal chromatographic resin from a few promising alternatives. The methodology starts with chromatographic modeling,parameters acquisition, and model validation, followed by model-based optimization of the chromatographic separation for the resins of interest.

pH-dependent uptake of fumaric acid in Saccharomyces cerevisiae under anaerobic conditions.

Microbial production of C(4) dicarboxylic acids from renewable resources has gained renewed interest. The yeast Saccharomyces cerevisiae is known as a robust microorganism and is able to grow at low pH, which makes it a suitable candidate for biological production of organic acids. However, a successful metabolic engineering approach for overproduction of organic acids requires an incorporation of a proper exporter to increase the productivity.

How to obtain true and accurate rate-values.

In metabolic flux calculations, the uptake and secretion rates (for substrate, O(2), CO(2), growth, (by)-products) are essential to arrive at correct calculated fluxes. Surprisingly, a lot of research has been published on the methods of flux calculations, but much less attention has been spent on the methods to obtain accurate and true uptake and secretion rates which are used as input. Therefore, this contribution focuses on.

Cytosolic NADPH balancing in Penicillium chrysogenum cultivated on mixtures of glucose and ethanol.

The in vivo flux through the oxidative branch of the pentose phosphate pathway (oxPPP) in Penicillium chrysogenum was determined during growth in glucose/ethanol carbon-limited chemostat cultures, at the same growth rate. Non-stationary (13)C flux analysis was used to measure the oxPPP flux. A nearly constant oxPPP flux was found for all glucose/ethanol ratios studied.

Genome-derived minimal metabolic models for Escherichia coli MG1655 with estimated in vivo respiratory ATP stoichiometry.

Metabolic network models describing growth of Escherichia coli on glucose, glycerol and acetate were derived from a genome scale model of E. coli. One of the uncertainties in the metabolic networks is the exact stoichiometry of energy generating and consuming processes.

Dynamic gene expression regulation model for growth and penicillin production in Penicillium chrysogenum.

As is often the case for microbial product formation, the penicillin production rate of Penicillium chrysogenum has been observed to be a function of the growth rate of the organism. The relation between the biomass specific rate of penicillin formation (q(p)) and growth rate (mu) has been measured under steady state conditions in carbon limited chemostats resulting in a steady state q(p)(mu) relation. Direct application of such a relation to predict the rate of product formation during dynamic conditions, as they occur, for example, in fed-batch experiments, leads to errors in the prediction, because q(p) is not an instantaneous function of the growth rate but rather lags behind because of adaptational and regulatory processes.

Metabolite and reaction inference based on enzyme specificities.

Motivation: Many enzymes are not absolutely specific, or even promiscuous: they can catalyze transformations of more compounds than the traditional ones as listed in, e.g. KEGG.

Model reduction and a priori kinetic parameter identifiability analysis using metabolome time series for metabolic reaction networks with linlog kinetics.

In this work, we present a time-scale analysis based model reduction and parameter identifiability analysis method for metabolic reaction networks. The method uses the information obtained from short term chemostat perturbation experiments. We approximate the time constant of each metabolite pool by their turn-over time and classify the pools accordingly into two groups: fast and slow pools.

Isotopic non-stationary 13C gluconate tracer method for accurate determination of the pentose phosphate pathway split-ratio in Penicillium chrysogenum.

Current (13)C labeling experiments for metabolic flux analysis (MFA) are mostly limited by either the requirement of isotopic steady state or the extremely high computational effort due to the size and complexity of large metabolic networks. The presented novel approach circumvents these limitations by applying the isotopic non-stationary approach to a local metabolic network. The procedure is demonstrated in a study of the pentose phosphate pathway (PPP) split-ratio of Penicillium chrysogenum in a penicillin-G producing chemostat-culture grown aerobically at a dilution rate of 0.

Metabolic flux and metabolic network analysis of Penicillium chrysogenum using 2D [13C, 1H] COSY NMR measurements and cumulative bondomer simulation.

At present two alternative methods are available for analyzing the fluxes in a metabolic network: (1) combining measurements of net conversion rates with a set of metabolite balances including the cofactor balances, or (2) leaving out the cofactor balances and fitting the resulting free fluxes to measured (13)C-labeling data. In this study these two approaches are applied to the fluxes in the glycolysis and pentose phosphate pathway of Penicillium chrysogenum growing on either ammonia or nitrate as the nitrogen source, which is expected to give different pentose phosphate pathway fluxes. The presented flux analyses are based on extensive sets of 2D [(13)C, (1)H] COSY data.

Cumulative bondomers: a new concept in flux analysis from 2D [13C,1H] COSY NMR data.

A well-established way of determining metabolic fluxes is to measure 2D [(13)C,(1)H] COSY NMR spectra of components of biomass grown on uniformly (13)C-labeled carbon sources. When using the entire set of measured data to simultaneously determine all fluxes in a proposed metabolic network model, the (13)C-labeling distribution in all measured compounds has to be simulated. This requires very large sets of isotopomer or cumomer balances.

A priori analysis of metabolic flux identifiability from (13)C-labeling data.

The (13)C-labeling technique was introduced in the field of metabolic engineering as a tool for determining fluxes that could not be found using the 'classical' method of flux balancing. An a priori flux identifiability analysis is required in order to determine whether a (13)C-labeling experiment allows the identification of all the fluxes. In this article, we propose a method for identifiability analysis that is based on the recently introduced 'cumomer' concept.

Deconvolution of light-scattering patterns by observing intensity fluctuations.

Results are presented on the statistical fluctuations occurring in a forward-light-scattering experiment to determine the particle size distribution. A sample of glass beads was measured using a Malvern 2600D instrument and analyzed with a proposed deconvolution procedure that incorporates the observed intensity fluctuations. This procedure yields a qualitative improvement of the solution, provides error intervals, and offers a better means for model discrimination.