Development of an on-line process monitoring for yeast cultivations via 2D-fluorescence spectroscopy

An optimum process is required in the field of food, pharmaceutical and biotechnological industry with the ultimate goal of achieving high productivity and high-quality products. In order to achieve this goal, there are many different parameters to be realized and controlled, e.g., physical, chemical and biological aspects of microbial bioprocesses. Microbial cultivations are a very complex process, therefore, reliable and efficient tools are required to receive as much real-time information for an on-line monitoring as possible, so that the processes can be controlled in time. The primary objective of this research was to apply a two-dimensional (2D) fluorescence spectroscopy to monitor glucose, ethanol and biomass concentrations of yeast cultivations. The measurement of one spectrum has 120 fluorescence intensity variables of excitation and emission wavelength combinations (WLCs) without consideration of the scattered light. To investigate which WLCs carry important and relevant information regarding the analyte concentrations, the three wavelength selection methods were implemented: a method based on loadings, variable importance in projection (VIP) and ant colony optimization. The five selected WLCs from each method for a particular analyte were evaluated by multiple linear regression (MLR) models. The selected WLCs, which showed the best predictive performance of the MLR models, were relevant to the analyte concentrations. Regarding the results of the MLR models, the most significant WLCs contained seven different excitation and emission wavelengths. They can be combined to have 38 WLCs for one spectrum based on the principle of fluorescence. They were in the area of NADH, tryptophan, pyridoxine, riboflavin and FAD/FMN. The 38 WLCs were used to predict the glucose, ethanol and biomass concentrations via partial least squares (PLS) regression. The best prediction from the PLS models with 38 WLCs had the percentage of root mean square error of prediction (pRMSEP) in the range of 3.1-6.3 %, which was not significantly different from the PLS models with the 120 variables. Therefore, the specific fluorescence sensor for yeast cultivations could be built with less filters, which would make it a low-cost device. The following plan of the research goal was to investigate the attribute of fluorophores inside cells in real time using a 2D fluorescence spectrometer. The considered intracellular fluorophores, such as NADH, tryptophan, pyridoxine, riboflavin and FAD/FMN were observed during the yeast cultivations under three different conditions: batch, fed-batch with the glucose pulse during a glucose growth phase (GP) and fed-batch with the glucose pulse during an ethanol growth phase (EP) after a diauxic shift. With the help of principal component analysis, the different states of the yeast cultivations, particularly the glucose pulse during EP, can be recognized and identified from the on-line fluorescence spectra. On the other hand, the change of the fluorescence spectra in the fed-batch process with the glucose pulse during GP was not recognizable. Remarkably, the intensities of the fluorophores due to the glucose pulse during EP did not change in the same direction. The fluorescence intensities of NADH and riboflavin increased, but the intensity of tryptophan, pyridoxine and FAD/FMN decreased. The conversion between tryptophan and NADH intensities was quantified as a proportional factor. It was calculated from the ratio of the area of NADH and tryptophan fluorescence intensity after the glucose addition until depletion. The proportional factor was independent on various glucose concentrations with the coefficient of determination, R2 = 0.999. The correlative intensity changes of these fluorophores demonstrate a metabolic switch from ethanol to glucose growth phase. Based on the previous experiments, a closed-loop control has been implemented for yeast cultivations. 2D fluorescence spectroscopy was applied for an on-line monitoring and control of yeast cultivations to attain pure oxidative metabolism. A glucose concentration is an important factor in a fed-batch process of Saccharomyces cerevisiae. Therefore, it has to be controlled under a critical concentration to avoid overflow metabolism and to gain high productivity of biomass. The characteristic of the NADH intensity can effectively identify the metabolic switch between oxidative and oxidoreductive states. Consequently, the feed rates were regulated using the NADH intensity as a metabolic signal. With this closed-loop control of the glucose concentration, a biomass yield was obtained at 0.5 gbiomass/gglucose. Additionally, ethanol production could be avoided during the controlled feeding phase. The fluorescence sensor with the signal of the NADH intensity has potential to control a glucose concentration under the critical value in real time. The experiments carried out show that 2D fluorescence spectroscopy has great potential in on-line monitoring and process control of the yeast cultivations. Consequently, it is promising to build up a compact and economical fluorescence sensor with the specific wavelengths using light-emitting diodes and photodiodes. The sensor would be a cost-effective and miniaturized device for routine analysis, which could be advantageous to real-time bioprocess monitoring.Im Bereich der Lebensmittel-, Pharma- und Biotech-Industrie ist ein optimaler Prozess mit dem Ziel einer hohen Produktivität und hohen Produktqualität erforderlich. Um dieses Ziel zu erreichen, sind viele verschiedene Parameter zu überwachen und zu regeln, z.B. physikalische, chemische und biologische Aspekte von mikrobiellen Bioprozessen. Kultivierung von Mikroorganismen ist ein komplexer Prozess, der für ein Online-Monitoring zuverlässige und effiziente Werkzeuge benötigt, um möglichst viele Informationen in Echtzeit zu erhalten, so dass eine Regelung realisiert werden kann. Das Hauptziel der Forschung war die Anwendung von 2D-Fluoreszenzspektroskopie zur Überwachung der Glukose-, Ethanol- und Biomassekonzentrationen von Hefekultivierung. Die Messung eines Spektrums besteht aus 120 Wellenlängenkombinationen (WLK) ohne Berücksichtigung des Streulichts. Um zu untersuchen, welche WLK wichtige und relevante Informationen über die Prozessgrößen enthalten, wurden drei Wellenlängenauswahlmethoden implementiert: Methode basierend auf Loadings, Variable Importance in Projection (VIP) und Ameisenkolonieoptimierung. Die fünf ausgewählten WLK jeder Methode für eine bestimmte Substanz wurden mit Hilfe der multilinearen Regression (MLR) bewertet. Die ausgewählten WLK, die die beste Vorhersageleistung des MLR-Modells zeigten, waren für die Prozessgrößen relevant. Bezüglich der Ergebnisse des MLR-Modells enthielten die wichtigsten WLK sieben verschiedene Anregungs- und Emissionswellenlängen. Basierend auf dem Prinzip der Fluoreszenz können sie zu 38 WLK für die Messung eines Teilspektrums kombiniert werden. Sie lagen im Bereich der Fluoreszenz von NADH, Tryptophan, Pyridoxin, Riboflavin und FAD/FMN. Diese 38 WLK wurden verwendet, um die Glukose-, Ethanol- und Biomassekonzentrationen über Partial Least Squares (PLS) Regression vorherzusagen. Die besten Vorhersagen der PLS-Modelle mit 38 WLK hatte relative Fehler im Bereich von 3,1-6,3 %. Das ist nicht signifikant schlechter als die PLS-Modellen mit 120 Variablen. Ein spezifischer Fluoreszenzsensor für die Hefekulturen könnte daher mit weniger Filtern gebaut werden, was ein kostengünstiges Gerät wäre. Forschungsziel war es, die Eigenschaften von Fluorophoren in den Zellen in Echtzeit mit einem 2D-Fluoreszenzspektrometer zu untersuchen. Die betrachteten intrazellulären Fluorophore wie NADH, Tryptophan, Pyridoxin, Riboflavin und FAD/FMN wurden während der Hefekultivierung unter drei verschiedenen Bedingungen beobachtet: Batch-Kultivierung, Fed-Batch-Kultivierung mit einem Glukose-Puls während der Glukosewachstumsphase (GP) und Fed-Batch-Kultivierung mit einem Glukose-Puls während der Ethanolwachstumsphase (EP) nach einer Diauxie. Mit Hilfe der Hauptkomponentenanalyse können die verschiedenen Zustände der Hefekultivierung, insbesondere der Glukose-Puls während des EP, aus den Online-Fluoreszenzspektren erkannt und identifiziert werden. Andererseits war die Änderung in den Fluoreszenzspektren der Fed-Batch-Kultivierung mit dem Glukose-Puls während der GP nicht erkennbar. Bemerkenswert war, dass sich die Intensitäten der Fluorophore durch den Glukose-Puls während des EP nicht alle in die gleiche Richtung verändert haben. Die Fluoreszenzintensitäten von NADH und Riboflavin nahmen zu, aber die Intensitäten von Tryptophan, Pyridoxin und FAD/FMN nahmen ab. Die Umwandlung von Tryptophan zu NADH konnte aufgezeigt und ein linearer Zusammenhang nachgewiesen werden. Der Proportionalfaktor war unabhängig von verschiedenen Glukosekonzentrationen mit einem Bestimmtheitsmaß von R2 = 0,999. Die korrelative Intensitätsänderung von den Fluorophoren zeigte die Stoffwechselveränderung von der Ethanol- zur Glukosewachstumsphase. Basierend auf den vorherigen Experimenten wurde eine Regelung für die Hefekultivierung implementiert. Die 2D-Fluoreszenzspektroskopie wurde zur Online-Überwachung und Kontrolle der Hefekultivierung eingesetzt, um einen reinen oxidativen Stoffwechsel zu erreichen. Die Höhe der Glukosekonzentration ist ein wichtiger Faktor in einem Fed-Batch-Prozess von Saccharomyces cerevisiae. Daher ist es notwendig die Glukosekonzentration unter einer kritischen Konzentration zu halten, um einen Überlaufstoffwechsel zu vermeiden und eine hohe Produktivität der Biomasse zu erreichen. Die Charakteristik der NADH-Intensität kann den metabolischen Wechsel zwischen oxidativen und oxidoreduktiven Zuständen effektiv identifizieren. Folglich wurde die Fütterungsrate auf Basis der Fluoreszenzintensität von NADH als Stoffwechselsignal geregelt. Mit dieser Regelung der Glukosekonzentration wurde ein Ausbeutekoeffizient von 0,5 gBiomass/gGlucose erzielt. Die Ethanolproduktion wurde so effektiv vermieden. Der Fluoreszenzsensor hat das Potenzial, die Glukosekonzentration unter dem kritischen Wert zu regeln. Die durchgeführten Experimente zeigen, dass die 2D-Fluoreszenzspektroskopie ein großes Potenzial in der Online-Überwachung und Prozesskontrolle hat. Daher ist es vielversprechend mit Hilfe von Leucht- und Photodioden einen kompakten und kostengünstigen Fluoreszenzsensor mit den spezifischen Wellenlängen aufzubauen. Der Sensor wäre ein preiswertes und miniaturisiertes Gerät für die Routineanalytik, was für die Online-Bioprozessüberwachung von Vorteil ist

Coordination and Control Mechanisms of Western European MNC towards Southeast Asia Subsidiaries: A Case Study of PZ Cussons

Abstract Date of final seminar:           June 3rd, 2010   Level: Master Thesis: EFO705 (15 Credits)   Authors: Ratiboon    Assawajaruwan (850522) Teeranuch Pumthong           (850508) Title: Coordination and Control Mechanisms of Western European MNC towards Southeast Asia Subsidiaries   Tutor: Leif Linnskog                         Examiner:       Ole     Liljefors   Research Problem:   “How does Western European MNC coordinate and control its subsidiaries in Southeast Asia?”   Purpose: To describe how Western European MNC controls and coordinates with its subsidiaries in Southeast Asia.   Method: The qualitative approach is employed for gathering data and analysis. The primary data is collected by the semi-structure interview. PZ Cussons is selected as a case study in order to study and describe the control and coordination mechanisms between headquarters and subsidiaries.   Conclusion: To achieve effective business operation and respond to increased complexity of international environments, it is important for a large and complex MNC as PZ Cussons to concern the control and coordination mechanisms between headquarters and subsidiaries. Considering different contexts among various subsidiaries, PZ Cussons structured the organization as multi-home-based MNC with partial in heterarchical management and deploys a combination of several control and coordination mechanisms to fit with each subsidiary’s environment. Transnational solution as well as control and coordination mechanisms as differentiated fit and shared value are integrated and implemented by PZ Cussons headquarters in UK to cooperate with Thailand and Indonesia subsidiaries in Southeast Asia.   Keywords: Multinational Company, Organization structure, Coordination mechanism

Feedback control based on NADH fluorescence intensity for Saccharomyces cerevisiae cultivations

Abstract Background A glucose concentration is an important factor for a fed-batch process of Saccharomyces cerevisiae. Therefore, it is necessary to be controlled under a critical concentration to avoid overflow metabolism and to gain high productivity of biomass. In the study, 2D fluorescence spectroscopy was applied for an online monitoring and controlling of the yeast cultivations to attain the pure oxidative metabolism. Results The characteristic of the NADH intensity can effectively identify the metabolic switch between oxidative and oxidoreductive states. Consequently, the feed rate was regulated using the single signal based on the fluorescence intensity of NADH. With this closed-loop control of the glucose concentration, a biomass yield was obtained at 0.5 g biomass/g glucose. In addition, ethanol production could be avoided during the controlled feeding phase. Conclusions The fluorescence sensor with a single signal of the NADH fluorescence intensity has potential to control a glucose concentration under the critical value in real time. Therefore, this achievement of the feedback control is promising to build up a compact and economical fluorescence sensor with the specific wavelength using light-emitting diodes and photodiodes. The sensor could be advantageous to the bioprocess monitoring because of a cost-effective and miniaturized device for routine analysis

Microbial Poly(hydroxybutyrate-co-hydroxyvalerate) Scaffold for Periodontal Tissue Engineering

In this study, we fabricated three dimensional (3D) porous scaffolds of poly(hydroxybutyrate-co-hydroxyvalerate) with 50% HV content. P(HB-50HV) was biosynthesized from bacteria Cupriavidus necator H16 and the in vitro proliferation of dental cells for tissue engineering application was evaluated. Comparisons were made with scaffolds prepared by poly(hydroxybutyrate) (PHB), poly(hydroxybutyrate-co-12%hydroxyvalerate) (P(HB-12HV)), and polycaprolactone (PCL). The water contact angle results indicated a hydrophobic character for all polymeric films. All fabricated scaffolds exhibited a high porosity of 90% with a sponge-like appearance. The P(HB-50HV) scaffolds were distinctively different in compressive modulus and was the material with the lowest stiffness among all scaffolds tested between the dry and wet conditions. The human gingival fibroblasts (HGFs) and periodontal ligament stem cells (PDLSCs) cultured onto the P(HB-50HV) scaffold adhered to the scaffold and exhibited the highest proliferation with a healthy morphology, demonstrating excellent cell compatibility with P(HB-50HV) scaffolds. These results indicate that the P(HB-50HV) scaffold could be applied as a biomaterial for periodontal tissue engineering and stem cell applications

Microbial Poly(hydroxybutyrate-co-hydroxyvalerate) Scaffold for Periodontal Tissue Engineering

In this study, we fabricated three dimensional (3D) porous scaffolds of poly(hydroxybutyrate-co-hydroxyvalerate) with 50% HV content. P(HB-50HV) was biosynthesized from bacteria Cupriavidus necator H16 and the in vitro proliferation of dental cells for tissue engineering application was evaluated. Comparisons were made with scaffolds prepared by poly(hydroxybutyrate) (PHB), poly(hydroxybutyrate-co-12%hydroxyvalerate) (P(HB-12HV)), and polycaprolactone (PCL). The water contact angle results indicated a hydrophobic character for all polymeric films. All fabricated scaffolds exhibited a high porosity of 90% with a sponge-like appearance. The P(HB-50HV) scaffolds were distinctively different in compressive modulus and was the material with the lowest stiffness among all scaffolds tested between the dry and wet conditions. The human gingival fibroblasts (HGFs) and periodontal ligament stem cells (PDLSCs) cultured onto the P(HB-50HV) scaffold adhered to the scaffold and exhibited the highest proliferation with a healthy morphology, demonstrating excellent cell compatibility with P(HB-50HV) scaffolds. These results indicate that the P(HB-50HV) scaffold could be applied as a biomaterial for periodontal tissue engineering and stem cell applications