A machine vision system for automated non-invasive assessment of cell viability via dark field microscopy, wavelet feature selection and classification

Wei N, Flaschel E, Friehs K, Nattkemper TW. A machine vision system for automated non-invasive assessment of cell viability via dark field microscopy, wavelet feature selection and classification. BMC Bioinformatics. 2008;9(1):449.Background: Cell viability is one of the basic properties indicating the physiological state of the cell, thus, it has long been one of the major considerations in biotechnological applications. Conventional methods for extracting information about cell viability usually need reagents to be applied on the targeted cells. These reagent-based techniques are reliable and versatile, however, some of them might be invasive and even toxic to the target cells. In support of automated noninvasive assessment of cell viability, a machine vision system has been developed. Results: This system is based on supervised learning technique. It learns from images of certain kinds of cell populations and trains some classifiers. These trained classifiers are then employed to evaluate the images of given cell populations obtained via dark field microscopy. Wavelet decomposition is performed on the cell images. Energy and entropy are computed for each wavelet subimage as features. A feature selection algorithm is implemented to achieve better performance. Correlation between the results from the machine vision system and commonly accepted gold standards becomes stronger if wavelet features are utilized. The best performance is achieved with a selected subset of wavelet features. Conclusion: The machine vision system based on dark field microscopy in conjugation with supervised machine learning and wavelet feature selection automates the cell viability assessment, and yields comparable results to commonly accepted methods. Wavelet features are found to be suitable to describe the discriminative properties of the live and dead cells in viability classification. According to the analysis, live cells exhibit morphologically more details and are intracellularly more organized than dead ones, which display more homogeneous and diffuse gray values throughout the cells. Feature selection increases the system's performance. The reason lies in the fact that feature selection plays a role of excluding redundant or misleading information that may be contained in the raw data, and leads to better results

Transesterification of phenylalanine by means of chymotrypsin in a continuous fixed bed reactor

An enzymic transesterification was carried out in a continuously operated fixed bed reactor. The reaction system consisted of immobilized a-chymotrypsin catalyzing the transfer of the L-phenylalanine radical from the racemic Pr ester to 1,4-butanediol, yielding L-phenylalanine 4-hydroxybutyl ester. The desired reaction was accompanied by alcoholysis due to the presence of PrOH liberated during the reaction and by hydrolysis of both the Pr and the hydroxybutyl ester. The problem of shifting pH during the reaction due to ester hydrolysis was overcome by adjusting the initial pH of the substrate feed soln. appropriately in order to obtain a sufficiently high buffer capacity provided by the free amino group of the esters. Thus, it was possible to work with shifting pH, and obvious disadvantage for operating reactors of low backmixing for this kind of reaction system. The overall reaction scheme was characterized by the appearance of a max. ester yield as a function of the operating time in case of batch reactors. Surprisingly, the yield became const. as a function of space-time for continuous operation due to a steeper pH drop. The max. productivity achieved with respect to the hydroxybutyl ester was .apprx.65 mol/L-day. [on SciFinder (R)

Perceiving molecular evolution processes in Escherichia coli by comprehensive metabolite and gene expression profiling

Transcript and metabolite abundance changes were analyzed in evolved and ancestor strains of Escherichia coli in three different evolutionary condition

Multiple model approach to modelling of Escherichia coli fed-batch cultivation extracellular production of bacterial phytase

The paper presents the implementation of multiple model approach to modelling of Escherichia coli BL21(DE3)pPhyt109 fed-batch cultivation processes for an extracellular production of bacterial phytase. Due to the complex metabolic pathways of microorganisms, the accurate modelling of bioprocesses is rather difficult. Multiple model approach is an alternative concept which helps in modelling and control of complex processes. The main idea is the development of a model based on simple submodels for the purposes of further high quality process control. The presented simulations of E. coli fed-batch cultivation show how the process could be divided into different functional states and how the model parameters could be obtained easily using genetic algorithms. The obtained results and model verification demonstrate the effectiveness of the applied concept of multiple model approach and of the proposed identification scheme. © 2007 by Pontificia Universidad Católica de Valparaíso

Antibiotic-free segregational plasmid stabilization in Escherichia coli owing to the knockout of triosephosphate isomerase (tpiA)

Selvamani RSV, Telaar M, Friehs K, Flaschel E. Antibiotic-free segregational plasmid stabilization in Escherichia coli owing to the knockout of triosephosphate isomerase (tpiA). Microbial Cell Factories. 2014;13(1): 58.Background: Segregational stability of plasmids is of major concern for recombinant bacterial production strains. One of the best strategies to counteract plasmid loss is the use of auxotrophic mutants which are complemented with the lacking gene along with the product-relevant ones. However, these knockout mutants often show unwanted growth in complex standard media or no growth at all under uncomplemented conditions. This led to the choice of a gene for knockout that only connects two essential arms of an essential metabolic pathway - the glycolysis. Results: Triosephosphate isomerase was chosen because its knockout will have a tremendous effect on growth on glucose as well as on glycerol. On glycerol the effect is almost absolute whereas on glucose growth is still possible, but with considerably lower rate than usual. This feature is essential because it may render cloning easier. This enzymatic activity was successfully tested as an alternative to antibiotic-based plasmid selection. Expression of a model recombinant beta-glucanase in continuous cultivation was possible with stable maintenance of the plasmid. In addition, the complementation of tpiA knockout strains by the corresponding plasmids and their growth characteristics were tested on a series of complex and synthetic media. The accumulation of methylglyoxal during the growth of tpiA-deficient strains was shown to be a possible cause for the growth disadvantage of these strains in comparison to the parent strain for the Keio Collection strain or the complemented knock-out strain. Conclusion: Through the use of this new auxotrophic complementation system, antibiotic-free cloning and selection of recombinant plasmid were possible. Continuous cultivation and recombinant protein expression with high segregational stability over an extended time period was also demonstrated

Multiple model approach to modelling of Escherichia coli fed-batch cultivation extracellular production of bacterial phytase

The paper presents the implementation of multiple model approach to modelling of Escherichia coli BL21(DE3)pPhyt109 fed-batch cultivation processes for an extracellular production of bacterial phytase. Due to the complex metabolic pathways of microorganisms, the accurate modelling of bioprocesses is rather difficult. Multiple model approach is an alternative concept which helps in modelling and control of complex processes. The main idea is the development of a model based on simple submodels for the purposes of further high quality process control. The presented simulations of E. coli fed-batch cultivation show how the process could be divided into different functional states and how the model parameters could be obtained easily using genetic algorithms. The obtained results and model verification demonstrate the effectiveness of the applied concept of multiple model approach and of the proposed identification scheme

Can we expect a breakthrough in downstream processing of biotechnological products in the near future?

Flaschel E. Can we expect a breakthrough in downstream processing of biotechnological products in the near future? Chemical Engineering & Technology. 2008;31(6):809

Reactor performance in the presence of catalyst deactivation

Flaschel E. Reactor performance in the presence of catalyst deactivation. In: Engineering in Life Sciences. ENGINEERING IN LIFE SCIENCES. Vol 6. Wiley-VCH; 2006: 131-138.The main variable of enzymatic processes is often found to be the operating temperature. An increase in temperature leads to higher rates for the catalytic transformation. However, beyond a certain temperature catalyst deactivation is winning the game. Therefore, processes should be optimized in order to determine the temperature which leads to a minimal demand of enzyme preparation. For the prediction of such optimal reactor operation, modeling of the temperature dependence of the process has to be performed. Examples of such modeling are given for the hydrolysis of lactose in UHT milk by means of three different beta-galactosidases - those from Aspergillus orvzae, Kluvveromyces lactis, and Escherichia coli. The reaction kinetics for a constant initial lactose concentration can be described by a model of two parameters, of which only one depends on temperature. For the lactase of E coli the reaction can be described as a simple reaction with first order kinetics. The deactivation mechanism includes a reversible as well as an irreversible path of denaturation. The temperature dependent parameters follow Arrheilius' and van't Hoff's law, respectively. On the basis of their particular reaction models all three enzymes can be compared with respect to their Optimum use. The models have been verified under laboratory conditions and have shown their usefulness for the prediction of optimum operating variables. Quite remarkable features have been found for the lactase of E. coli

Renovation of biotechnological products - Faces a revolution?

Flaschel E. Renovation of biotechnological products - Faces a revolution? CHEMIE INGENIEUR TECHNIK. 2008;80(6):695

Biocatalysis - New tasks, new approaches

Flaschel E. Biocatalysis - New tasks, new approaches. CHEMIE INGENIEUR TECHNIK. 2010;82(1-2):3