An in situ probe for on-line monitoring of cell density and viability on the basis of dark field microscopy in conjunction with image processing and supervised machine learning

Fermentation industries would benefit from on-line monitoring of important parameters describing cell growth such as cell density and viability during fermentation processes. For this purpose, an in situ probe has been developed, which utilizes a dark field illumination unit to obtain high contrast images with an integrated CCD camera. To test the probe, brewer's yeast Saccharomyces cerevisiae is chosen as the target microorganism. Images of the yeast cells in the bioreactors are captured, processed, and analyzed automatically by means of mechatronics, image processing, and machine learning. Two support vector machine based classifiers are used for separating cells from background, and for distinguishing live from dead cells afterwards. The evaluation of the in situ experiments showed strong correlation between results obtained by the probe and those by widely accepted standard methods. Thus, the in situ probe has been proved to be a feasible device for on-line monitoring of both cell density and viability with high accuracy and stability.     

On-line stoichiometry and identification of metabolic state under dynamic process conditions

A method for the on-line calculation of conversion rates and yield coefficients under dynamic process conditions was developed. The method is based on cumulated mass balances using a moving average method. Elemental balances were used to test the measured cumulated quantities for gross errors and inappropriate stoichiometry definition followed by data reconciliation and estimation of non-measured conversion rates, using a bioprocess set-up including multiple on-line analysis techniques. The quantitative potential of the proposed method is demonstrated by executing transient experiments in aerobic cultures of Saccharomyces cerevisiae on glucose. Rates and yield coefficients could be consistently quantified in shift-up, shift-down, and accelerostat experiments. The method shows the capability to describe quantitatively transient changes in metabolism including uncoupling of catabolism and anabolism, also for the case when multiple components of metabolism are not measured. The validity of the experiment can be evaluated on-line. Additionally, the method detects with high sensitivity inappropriate stoichiometry definition, such as a change in state of metabolism. It was shown that concentration values can be misleading for the identification of the metabolic state. In contrast, the proposed method provides a clear picture of the metabolic state and new physiological regulations could be revealed. Hence, the novelty of the proposed method is the on-line availability of consistent stoichiometric coefficients allowing a significant speed up in strain characterization and bioprocess development using minimal knowledge of the metabolism. Additionally, it opens up the use of transient experiments for physiological studies.     

Metallic oxide CdIn2O4 films for the label free electrochemical detection of DNA hybridization

DNA functionalised semiconductor metallic oxide electrodes have been developed for the direct electrochemical detection of DNA hybridization, without labelling or the introduction of a redox couple. Conductive CdIn(2)O(4) thin films with controlled properties were deposited on glass substrates using an aerosol pyrolysis technique. The films exhibit a polycrystalline microstructure with a surface roughness of 1.5 nm (r.m.s.) and an electrical resistivity ranging between 1 and 3 x 10(-3) Omega cm. These electrodes were functionalised using hydroxylation and silanisation steps, to allow the binding of DNA probe sequences (20 bases). The electrical detection of DNA hybridization with complementary sequences has been performed using electrochemical impedance spectrometry (EIS) measuring the variation of impedance before and after hybridization. Two set-ups were used, a standard set-up including three electrodes and a set-up including two symmetrical electrodes. In both configurations, a significant increase of the impedance modulus, more particularly of the real part of the impedance (160-225% according to the electrochemical cell used) has been obtained over a frequency range of 10-10(5)Hz. DNA hybridization has also been systematically confirmed using the fluorescence spectrometry. This study emphasizes the high sensitivity of the CdIn(2)O(4) as a working electrode for the detection of biological events occurring at the electrode surface.     

High-cell-density fermentation of recombinant Saccharomyces cerevisiae using glycerol

To obtain a high cell density of recombinant Saccharomyces cerevisiae (INVSc 1 strain bearing a 2 microm plasmid, pYES2 containing a GAL1 promoter for expression of the beta-galactosidase gene), the yeast was grown with glycerol as the substrate by fed-batch fermentation. The feeding strategy was based on an on-line response of the medium pH to the consumption of glycerol. The approach was to feed excess carbon into the medium to create a benign environment for rapid biomass buildup. During cell growth in the presence of glycerol, the release of protons in the medium caused a decrease in pH and the consumption rate of ammonium phosphate served as an on-line indicator for the metabolic rate of the organism. The extent of glycerol feeding in a fed-batch mode with pH control at 5.0 +/- 0.1 was ascertained from the automatic addition of ammonium phosphate to the medium. The glycerol feeding to ammonium phosphate addition ratio was found to be 2.5-3.0. On the basis of the experiments, a maximum dry cell biomass of 140 g per liter and a productivity of 5.5 g DCW/L/h were achieved. The high cell density of S. cerevisiae obtained with good plasmid stability suggested a simple and efficient fermentation protocol for recombinant protein production.     

On-line biomass monitoring by capacitance measurement.

An in-situ, steam-sterilizable capacitance probe was used to follow the biomass concentration on-line, in bioreactors from 20 to 2000 l total volume. Microbial cultures of Saccharomyces cerevisiae, Pichia pastoris and Streptomyces virginiae were grown in batch and fed-batch culture in both defined and complex media in order to demonstrate the wide dynamic operating range of the instrument. A linear correlation was found between the on-line capacitance measurement and the off-line measurements (optical density, OD620; packed mycelial volume, PMV; biomass concentration X, and colony forming units, CFU ml-1) for biomass concentrations (dry cell weight) up to 30 g l-1 (St. virginiae), 106 g l-1 (S. cerevisiae) and 89 g l-1 (P. pastoris). The on-line capacitance measurement was slightly influenced by variations in agitation speed and strong extraneous radio frequencies. A specific capacitance constant (Cs) was defined for all microbial cells which was dependent on cell viability and cell size. The Cs was easy to calculate using the on-line capacitance measurement and an off-line estimation of biomass concentration. The Biomass Monitor proved suitable for precise on-line monitoring of both homogeneous (uni-cellular) and heterogeneous (mycelial) cultures in bioreactors.     

Detection of microbial concentration in ice-cream using the impedance technique

The detection of microbial concentration, essential for safe and high quality food products, is traditionally made with the plate count technique, that is reliable, but also slow and not easily realized in the automatic form, as required for direct use in industrial machines. To this purpose, the method based on impedance measurements represents an attractive alternative since it can produce results in about 10 h, instead of the 24–48 h needed by standard plate counts and can be easily realized in automatic form. In this paper such a method has been experimentally studied in the case of ice-cream products. In particular, all main ice-cream compositions of real interest have been considered and no nutrient media has been used to dilute the samples. A measurement set-up has been realized using benchtop instruments for impedance measurements on samples whose bacteria concentration was independently measured by means of standard plate counts. The obtained results clearly indicate that impedance measurement represents a feasible and reliable technique to detect total microbial concentration in ice-cream, suitable to be implemented as an embedded system for industrial machines.     

Ethanol fermentation in an immobilized cell reactor using Saccharomyces cerevisiae

Fermentation of sugar by Saccharomyces cerevisiae, for production of ethanol in an immobilized cell reactor (ICR) was successfully carried out to improve the performance of the fermentation process. The fermentation set-up was comprised of a column packed with beads of immobilized cells. The immobilization of S. cerevisiae was simply performed by the enriched cells cultured media harvested at exponential growth phase. The fixed cell loaded ICR was carried out at initial stage of operation and the cell was entrapped by calcium alginate. The production of ethanol was steady after 24 h of operation. The concentration of ethanol was affected by the media flow rates and residence time distribution from 2 to 7 h. In addition, batch fermentation was carried out with 50 g/l glucose concentration. Subsequently, the ethanol productions and the reactor productivities of batch fermentation and immobilized cells were compared. In batch fermentation, sugar consumption and ethanol production obtained were 99.6% and 12.5% v/v after 27 h while in the ICR, 88.2% and 16.7% v/v were obtained with 6 h retention time. Nearly 5% ethanol production was achieved with high glucose concentration (150 g/l) at 6 h retention time. A yield of 38% was obtained with 150 g/l glucose. The yield was improved approximately 27% on ICR and a 24 h fermentation time was reduced to 7 h. The cell growth rate was based on the Monod rate equation. The kinetic constants (K(s) and mu(m)) of batch fermentation were 2.3 g/l and 0.35 g/lh, respectively. The maximum yield of biomass on substrate (Y(X-S)) and the maximum yield of product on substrate (Y(P-S)) in batch fermentations were 50.8% and 31.2% respectively. Productivity of the ICR were 1.3, 2.3, and 2.8 g/lh for 25, 35, 50 g/l of glucose concentration, respectively. The productivity of ethanol in batch fermentation with 50 g/l glucose was calculated as 0.29 g/lh. Maximum production of ethanol in ICR when compared to batch reactor has shown to increase approximately 10-fold. The performance of the two reactors was compared and a respective rate model was proposed. The present research has shown that high sugar concentration (150 g/l) in the ICR column was successfully converted to ethanol. The achieved results in ICR with high substrate concentration are promising for scale up operation. The proposed model can be used to design a lager scale ICR column for production of high ethanol concentration.     

Achiral-chiral LC/LC-FLD coupling for determination of carvedilol in plasma samples for bioequivalence purposes

Bioequivalence data for two pharmaceutical formulations (solid oral dosage forms) containing carvedilol is presented for both racemic and enantiomers of the active substance. This was achieved by on-line coupling of two liquid chromatographic separations followed by fluorescence detection. The first LC dimension was used for a fast separation of racemic carvedilol from propranolol (IS) and the endogenous matrix, by means of a reversed phase mechanism. The peak of racemic carvedilol was on-line transferred to the second enantioselective LC dimension, based on a reversed phase separation on cellulose tris(3,5-dimethyl-phenylcarbamate) stationary phase. Both stages were monitored over a single run by means of a fluorescence detector operated at an excitation wavelength of 285 nm and an emission wavelength of 355 nm. Automated shortcutting of the racemic carvedilol peak to the chiral column and simultaneous detection over the two LC dimensions have been obtained by using an experimental set-up based on two six-port rotative switching valves. Linearity was demonstrated on the interval 2-150 ng/mL for racemic carvedilol and on 1-75 ng/mL intervals for enantiomers. LLOQ fits between 0.7 and 1.4 ng/mL. Recoveries of the target compounds are 87+/-4 and 81+/-4% for the IS. Precision ranged from 0.6 to 2.5% and the mean accuracy obtained on quality control samples (measured as % bias) over the whole study falls between -0.8 and 6.3%.     

On-line cell mass monitoring of Saccharomyces cerevisiae cultivations by multi-wavelength fluorescence

The catalyst in bioprocesses, i.e. the cell mass, is one of the most challenging and important variables to monitor in bioprocesses. In the present study, cell mass in cultivations with Saccharomyces cerevisiae was monitored on-line with a non-invasive in situ placed sensor measuring multi-wavelength culture fluorescence. The excitation wavelength ranged from 270 to 550 nm with 20 nm steps and the emission wavelength range was from 310 to 590 nm also with 20 nm steps. The obtained spectra were analysed chemometrically with the multi-way technique, parallel factor analysis (PARAFAC), resulting in a decomposition of the multivariate fluorescent landscape, whereby underlying spectra of the individual intrinsic fluorophors present in the cell mass were estimated. Furthermore, gravimetrically determined cell mass concentration was used together with the fluorescence spectra for calibration and validation of multivariate partial least squares (PLS) regression models. Both two- and three-way models were calculated, the models behaved similarly giving root mean square error of prediction (RMSEPs) of 0.20 and 0.19 g l(-1), respectively, when test set validation was used. Based on this work, it is evident that on-line monitoring of culture fluorescence can be used for estimation of the cell mass concentration during cultivations.     

Effectiveness of the No action level protocol for head & neck patients – Time considerations

BackgroundThe No Action Protocol (NAL) was used to diminish the systematic set-up error. Recently, owing to the development of image registration technologies, the on-line positioning control is more often used. This method significantly reduces the CTV–PTV margin at the expense of the lengthening of a treatment session. The efficiency of NAL in decreasing the total treatment time for Head&Neck patients was investigated.MethodsResults of set-up control of 30 patients were analyzed. The set-up control was carried out on-line. For each patient and each fraction, the set-error and the time needed for making the set-up control procedure were measured. Next, retrospectively, the NAL was applied to this data. The number of initial errors (without interventions) and after NAL protocol were compared in terms of errors larger than 3 and 4 mm. The average and total time used for portal control was calculated and compared.ResultsThe number of setup errors in the posterior-anterior, inferior-superior, and right–left directions ≥3 mm and ≥4 mm were 98, 79, and 91 sessions and 44, 38 and 30 sessions out of 884 sessions. After NAL protocol the number of errors ≥3 mm and ≥4 mm decreased to 84, 57, and 39 sessions and 31, 15 and 10 sessions, respectively. The average time needed for one set-up control was 5.1 min. NAL protocol allows saving 4049 min for the whole group.ConclusionsFor locations where the random set-up errors are small, the NAL enables a very precise treatment of patients. Implementation of this protocol significantly decreases the total treatment time.     

Flow injection analysis-based methodology for automatic on-line monitoring and quality control for biodiesel production

An automated on-line approach based on determination of free and bound glycerol was here proposed to monitor biodiesel production. The method was based on liquid-liquid extraction of glycerol from the biodiesel to an aqueous ethanolic phase in which glycerol is oxidized to formaldehyde with meta periodate with subsequent reaction with acetylacetone. The reaction product was photometrically measured at 410 nm. Free and bound glycerol were differentiated by glycerides hydrolysis with potassium ethylate. The experimental set-up consisted of a flow-injection manifold for liquid-liquid extraction without phase separation and iterative change of the flow direction that enabled: (a) filling the flow manifold with a meta periodate-acetylacetone acceptor phase; (b) sampling of small amounts (microl) from the reactor; (c) determination of free glycerol by extraction from biodiesel to the aqueous phase with simultaneous oxidation-reaction with acetylacetone in the acceptor phase; (d) continuous monitoring of the aqueous phase by passage through a photometric detector; (e) filling the flow manifold with a potassium ethylate-meta periodate-acetylacetone new acceptor phase; (d) repetition of steps b-to-d to determine total glycerol after saponification of the bound glycerol by potassium ethylate; and (f) determination of bound glycerol by difference between the second and first analyses. The results showed that the proposed automated on-line method is a suitable option in routine analysis during biodiesel production.     

A composite sensor array impedentiometric electronic tongue Part II. Discrimination of basic tastes

An impedentiometric electronic tongue based on the combination of a composite sensor array and chemometric techniques aimed at the discrimination of soluble compounds able to elicit different gustative perceptions is presented. A composite array consisting of chemo-sensitive layers based on carbon nanotubes or carbon black dispersed in polymeric matrices and doped polythiophenes was used. The electrical impedance of the sensor array was measured at a frequency of 150 Hz by means of an impedance meter. The experimental set-up was designed in order to allow the automatic selection of a test solution and dipping of the sensor array following a dedicated measurement protocol. Measurements were carried out on 15 different solutions eliciting 5 different tastes (sodium chloride, citric acid, glucose, glutamic acid and sodium dehydrocholate for salty, sour, sweet, umami and bitter, respectively) at 3 concentration levels comprising the human perceptive range. In order to avoid over-fitting, more than 100 repetitions for each sample were carried in a 4-month period. Principal component analysis (PCA) was used to detect and remove outliers. Classification was performed by linear discriminant analysis (LDA). A fairly good degree of discrimination was obtained.     

High density culture of mammalian cells with dynamic perfusion based on on-line oxygen uptake rate measurements

In a continuous culture with cell retention the perfusion rate must be adjusted dynamically to meet the cellular demand. An automated mechanism of adjusting the perfusion rate based on real-time measurement of the metabolic load of the bioreactor is important in achieving a high cell concentration and maintaining high viability. We employed oxygen uptake rate (OUR) measurement as an on-line metabolic indicator of the physiological state of the cells in the bioreactor and adjusted the perfusion rate accordingly. Using an internal hollow fiber microfiltration system for total cell retention, a cell concentration of almost 10⁸ cells/mL was achieved. Although some aggregates were formed during the cultivation, the viability remained high as examined with confocal microscopy after fluorescent vital staining. The results demonstrate that on-line OUR measurement facilitates automated dynamic perfusion and allows a high cell concentration to be achieved.     

Assessment of cytotoxicity by impedance spectroscopy

This paper describes a simple and convenient method to monitor on-line cell adhesion by electrical impedance measurements. Immortalized mouse fibroblasts, BALB/3T3, were cultured onto interdigitated electrode structures integrated into the bottom of an in-house fabricated device. Impedance modulus, phase, real and imaginary parts were considered separately and plotted as function of frequency and time to better understand and select the component giving more information on cell adhesion changes. For cytotoxicity assessment, the cells were treated with different concentrations of sodium arsenite used as model toxicant and their responses were monitored on-line. The half inhibition concentration, the required concentration to achieve 50% inhibition, derived from the measurements fall between the results obtained using standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test and colony forming efficiency assay confirming the good sensitivity of the system. In term of impedance signal, the modulus results was found to be the most sensitive of the considered components for cytotoxicity testing of chemicals.     

Comparison of viable cell concentration estimation methods for a mammalian cell cultivation process

Various mechanistic and black-box models were applied for on-line estimations of viable cell concentrations in fed-batch cultivation processes for CHO cells. Data from six fed-batch cultivation experiments were used to identify the underlying models and further six independent data sets were used to determine the performance of the estimators. The performances were quantified by means of the root mean square error (RMSE) between the estimates and the corresponding off-line measured validation data sets. It is shown that even simple techniques based on empirical and linear model approaches provide a fairly good on-line estimation performance. Best results with respect to the validation data sets were obtained with hybrid models, multivariate linear regression technique and support vector regression. Hybrid models provide additional important information about the specific cellular growth rates during the cultivation.     

On-line monitoring of marine cyanobacterial cultivation based on phycocyanin fluorescence.

A novel on-line fluorescence monitoring system for marine cyanobacterial cultivation was developed. This method is based on the measurement of intracellular phycocyanin content, which is the major light harvesting protein. A fluorescence spectrophotometer, equipped with a flow cell connected with a culture liquid recycling tube was used. Experiments were carried out using a marine unicellular cyanobacteria Synechococcus sp. NKBG 042902 isolated from Japanese coastal sea water. We have optimized excitation wavelength to avoid the light scattering, using non-pigmented old cells which no longer contained phycocyanin. At an excitation wavelength of 590 nm, light scattering was minimized. Viable cell concentration could be measured in the range of 2 x 10(6) to 2 x 10(8) cells per ml, without pronounced light scattering. Continuous monitoring of marine cyanobacteria cultivation was performed. Cell concentrations were determined by both culture fluorescence and by using a hemacytometer. A good linear correlation was obtained. We conclude that on-line monitoring of cyanobacterial culture fluorescence based on phycocyanin is a rapid, efficient and also versatile method for determining viable cell concentration.     

High viable cell concentration fed-batch cultures of hybridoma cells through on-line nutrient feeding

A hybridoma cell line was cultivated in fed-batch cultures using a low-protein, serum-free medium. On-line oxygen uptake rate (OUR) measurement was used to adjust the nutrient feeding rate based on glucose consumption, which was estimated on-line using the stoichiometric relations between glucose and oxygen consumption. Through on-line control of the nutrient feeding rate, not only sufficients were supplied for cell growth and antibody production, but also the concentrations of glucose and other important nutrients such as amino acids were maintained at low levels during the cell growth phase. During the cultivation, cell metabolism changed from high lactate production and low oxygen consumption to low lactate production and high oxygen consumption. As a result the accumulation of lactate was reduced and the growth phase was extended. In comparison with the batch cultures, in which cells reached a concentration of approximately 2 x 10(6) cells/mL, a very high concentration of 1.36 x 10(7) cells/mL with a high cell viability (>90%) was achieved in the fed-batch culture. By considering the consumption of glucose and amino acids, as well as the production of cell mass, metabolites, and antibodies, a well-closed material balance was established. Our results demonstrate the value of coupling on-line OUR measurement and the stoichiometric realations for dynamic nutrient feeding in high cell concentration fed batch cultures. (c) 1995 John Wiley & Sons, Inc.     

Development of a new rapid measurement technique for fish embryo membrane permeability studies using impedance spectroscopy

Information on fish embryo membrane permeability is vital in their cryopreservation. Whilst conventional volumetric measurement based assessment methods have been widely used in fish embryo membrane permeability studies, they are lengthy and reduce the capacity for multi-embryo measurement during an experimental run. A new rapid 'real-time' measurement technique is required to determine membrane permeability during cryoprotectant treatment. In this study, zebrafish (Danio rerio) embryo membrane permeability to cryoprotectants was investigated using impedance spectroscopy. An embryo holding cell, capable of holding up to 10 zebrafish embryos was built incorporating the original system electrods for measuring the impedance spectra. The holding cell was tested with deionised water and a series of KCl solutions with known conductance values to confirm the performance of the modified system. Untreated intact embryos were then tested to optimise the loading capacity and sensitivity of the system. To study the impedance changes of zebrafish embryos during cryoprotectant exposure, three, six or nine embryos at 50% epiboly stage were loaded into the holding cell in egg water, which was then removed and replaced by 0.5, 1.0, 2.0 or 3M methanol or dimethyl sulfoxide (DMSO). The impedance changes of the loaded embryos in different cryoprotectant solutions were monitored over 30 min at 22 degrees C, immediately following embryo exposure to cryoprotectants, at the frequency range of 10-10(6)Hz. The impedance changes of the embryos in egg water were used as controls. Results from this study showed that the optimum embryo loading level was six embryos per cell for each experimental run. The optimum frequency was identified at 10(3.14) or 1,380 Hz which provided good sensitivity and reproducibility. Significant impedance changes were detected after embryos were exposed to different concentrations of cryoprotectants. The results agreed well with those obtained from conventional volumetric based studies.     

On-line determination of glucose in biotechnological processes: comparison between FIA and an in situ enzyme electrode.

Two different analysis techniques for on-line monitoring of glucose in biotechnological processes have been tested: an in situ enzyme electrode and a flow injection analysis system (FIA). The measuring ranges, detection limits, response times and the reliabilities of each system have been compared during monitoring of batch and continuous cultures of Saccharomyces cerevisiae.     

On-line monitoring of monoclonal antibody formation in high density perfusion culture using FIA

An automated flow injection system for on-line analysis of proteins in real fermentation fluids was developed by combining the principles of stopped-flow, merging zones flow injection analysis (FIA) with antigen-antibody reactions. IgG in the sample reacted with its corresponding antibody (a-IgG) in the reagent solution. Formation of insoluble immunocomplexes resulted in an increase of the turbidity which was determined photometrically. This system was used to monitor monoclonal antibody production in high cell density perfusion culture of hybridoma cells. Perfusion was performed with a newly developed static filtration unit equipped with hydrophilic microporous tubular membranes. Different sampling devices were tested to obtain a cell-free sample stream for on-line product anlysis of high molecular weight (e.g., monoclonal antibodies) and low molecular weight (e.g., glucose, lactate) medium components. In fermentation fluids a good correlation (coefficient: 0.996) between the FIA method and an ELISA test was demonstrated. In a high density perfusion cultivation process mAb formation was succesfully monitored on-line over a period of 400 h using a reliable sampling system. Glucose and lactate were measured over the same period of time using a commercially available automatic analyser based on immobilized enzyme technology.Abbreviations TIA Turbidimetric immunoassay - mAb Monoclonal Antibody