Bioprocess Monitoring and Control in Pseudomonas cepacia and recombinant Escherichia coli cultivations

The control of cultivation conditions and its influence on growth and production have been investigated. The use of fed-batch processes have been demonstrated for several cultivation systems and so has the use of different control signals in these systems. Fed-batch cultivation of Pseudomonas cepacia with on-line measurement and control of the growth rate limiting (toxic) substrate, sodium-salicylate, was investigated. The use of an external filter unit made it possible to determine the substrate level on-line, in the bioreactor. The measured signal was used in a control-loop for manipulating the feed-flow rate around a pre-determined feed profile. Conventional analytical process equipment (dissolved oxygen sensor) have been used in a novel way to gain information on the metabolic status of Escherichia coli cells in glucose limited fed-batch cultivations. Models were constructed and compared to experimental results. It was possible to detect acetic acid formation from responses in dissolved oxygen signal upon transient pulses in glucose feed rate. The influence of medium composition have been investigated in recombinant Escherichia coli protein production (xylanase deletion derivatives from Rhodothermus marinus). Using an exponential increase in feed-flow rate, resulting in a specific growth rate below µmax, the substrate level (glucose) could be kept at a low level in the bioreactor. The use of controlled cultivation conditions have made it possible to use lactose (an inexpensive alternative to IPTG) to induce the T7lac promoter for recombinant protein production. The impact of recombinant protein production (protein L), using the temperature induced lambdaPR promotor, on growth rate and viability, was investigated and compared to the response of a non recombinant strain. Monitoring of acetic acid using flow injection analysis was carried out. The stability of the immobilised enzymes, acetate kinase and pyruvate kinase, was very low when monitoring in cultivation systems. The presence of pyruvate in the medium interfered in the analytical system. That was solved by simultaneously measuring pyruvate and subtracting its contribution from the measured acetate signal. An integrated analytical system for on-line measurements of intracellular compounds was developed. It allows measurement in cell debris containing samples by use of expanded beds for enzyme immobilisation in the analytical system

Utformning av användarutbildning vid implementering av ERP-system

Enterprise resource planning (ERP) system används idag för att sammankoppla företags informationsflöden. Implementering av dessa system har över tid visat sig vara utmanande där bland annat utbildning utgör en viktig del i att detta arbete sker framgångsrikt. Med hjälp av en fallstudie har en mjukvaruleverantörs arbete med att utbilda kunder vid implementering av ERP-system studerats. Studien syftar till att kartlägga utbildningsprocessen för att undersöka hur utbildning i samband med systemimplementering kan utformas för att stötta kunders arbete med att nå sina målsättningar. Tre metoder för datainsamling har använts, samtliga med en kvalitativ ansats. Semistrukturerade intervjuer har genomförts med konsulter, konsultchef och kund. Dokumentgranskning har genomförts för att undersöka de utbildningsmaterial de idag erbjuder kunder och observation har använts för att ge insikt i hur ett utbildningstillfälle ser ut. Studien visar på kunskapsbrister hos kunder i den initiala fasen samt att det finns ett behov av att erbjuda ett interaktivt utbildningsmaterial och det behövs ett säkerställande av kunders kunskap innan driftstart. För att hantera detta föreslår resultatet en uppstart med hjälp av gamification och ett systematiskt sätt att tillhandahålla interaktivt utbildningsmaterial samt ett kunskapstest inför driftstart.The use of enterprise resource planning (ERP) systems is a common way to access information throughout different parts of a company. Implementation of these systems has shown to be challenging. One of the key elements towards a successful implementation has proven to be user training. Through this case study a software vendor has been examined on how user training is conducted during the implementation of an ERP-system. The study aims to investigate how user training can be conducted in order to support customer objectives. Three methods of data collection have been used, all with a qualitative approach. These are as follows, semi-structured interviews with consultants, manager and customer. Document examination to evaluate the currently used training materials and observation to gain insight in the actual training process. Findings show that customers have a lack of knowledge in the initial phase and that there is a need for interactive user training material as well as a way to ensure user knowledge prior to launch. To ensure this the study suggests providing initial user training through gamification, a systematic way of providing interactive user training material and a knowledge test of maneuvering the system prior to launch

Utformning av användarutbildning vid implementering av ERP-system

Enterprise resource planning (ERP) system används idag för att sammankoppla företags informationsflöden. Implementering av dessa system har över tid visat sig vara utmanande där bland annat utbildning utgör en viktig del i att detta arbete sker framgångsrikt. Med hjälp av en fallstudie har en mjukvaruleverantörs arbete med att utbilda kunder vid implementering av ERP-system studerats. Studien syftar till att kartlägga utbildningsprocessen för att undersöka hur utbildning i samband med systemimplementering kan utformas för att stötta kunders arbete med att nå sina målsättningar. Tre metoder för datainsamling har använts, samtliga med en kvalitativ ansats. Semistrukturerade intervjuer har genomförts med konsulter, konsultchef och kund. Dokumentgranskning har genomförts för att undersöka de utbildningsmaterial de idag erbjuder kunder och observation har använts för att ge insikt i hur ett utbildningstillfälle ser ut. Studien visar på kunskapsbrister hos kunder i den initiala fasen samt att det finns ett behov av att erbjuda ett interaktivt utbildningsmaterial och det behövs ett säkerställande av kunders kunskap innan driftstart. För att hantera detta föreslår resultatet en uppstart med hjälp av gamification och ett systematiskt sätt att tillhandahålla interaktivt utbildningsmaterial samt ett kunskapstest inför driftstart.The use of enterprise resource planning (ERP) systems is a common way to access information throughout different parts of a company. Implementation of these systems has shown to be challenging. One of the key elements towards a successful implementation has proven to be user training. Through this case study a software vendor has been examined on how user training is conducted during the implementation of an ERP-system. The study aims to investigate how user training can be conducted in order to support customer objectives. Three methods of data collection have been used, all with a qualitative approach. These are as follows, semi-structured interviews with consultants, manager and customer. Document examination to evaluate the currently used training materials and observation to gain insight in the actual training process. Findings show that customers have a lack of knowledge in the initial phase and that there is a need for interactive user training material as well as a way to ensure user knowledge prior to launch. To ensure this the study suggests providing initial user training through gamification, a systematic way of providing interactive user training material and a knowledge test of maneuvering the system prior to launch

Efficient production of truncated thermostable xylanases from Rhodothermus marinus in Escherichia coli fed-batch cultures

A cultivation strategy for the production of two truncated thermostable recombinant xylanases (Xyn1ΔN and Xyn1ΔNC) was developed. Fed-batch cultivations of Escherichia coli strain BL21(DE3) with a controlled exponential glucose feed led to high specific production of the recombinant proteins. Addition of complex nutrients (e.g. Tryptone Soya Broth (TSB)) to the media were shown to increase both the specific growth rate during the production phase and the production per cell. The final cellmass concentration depended on the time of induction in relation to both the feed- start and the expected time at which the cultivation had to be terminated due to oxygen transfer limitations or cell lysis. The gene used for the genetic constructions (encoding Xyn1ΔN and Xyn1ΔNC) was originally isolated from Rhodothermus marinus. Recombinant protein expression was controlled by the T7 lac-promoter and induced in the fed-batch phase at low glucose concentrations by the single addition of either lactose or isopropyl-thio-β-D-galactoside (IPTG). In lactose-induced cells, the production of recombinant xylanase was delayed for approximately 30 min in comparison with those induced with IPTG, but the specific product levels were comparable at 3 h after induction. At this time, approximately 35% of the intracellular protein content was constituted by recombinant xylanase. Under the cultivation conditions used, production of the shorter deletion derivative (Xyn1ΔNC) led to nonspecific leakage and cell lysis, starting 1.5 or 2 h after induction with IPTG or lactose, respectively. At 3 h after induction, 50% of the produced protein (Xyn1ΔNC) was found in the culture medium. This was not the case for the longer protein (Xyn1ΔN), where only 10% of the xylanase leaked into the medium

Acetate formation and dissolved oxygen responses to feed transients in Escherichia coli fermentations: Modeling and experiments

Two hypotheses for acetate formation in Escherichia coli are used to obtain relations between glucose uptake, oxygen uptake and acetate production. Based on these relations, models for dissolved oxygen responses to feed rate transients are derived. Simulations of the models are compared with experimental data. It is shown that information about acetate production can be obtained from the dissolved oxygen measurement by making short pulses in the feed rate

On-line Detection of Acetate Formation in Escherichia coli Cultures using Dissolved Oxygen Responses to Feed Transients

Recombinant protein production in Escherichia coli can be significantly reduced by acetate accumulation. It is demonstrated that acetate production can be detected on-line with a standard dissolved oxygen sensor by superimposing short pulses to the substrate feed rate. Assuming that acetate formation is linked to a respiratory limitation, a model for dissolved oxygen responses to transients in substrate feed rate is derived. The model predicts a clear change in the character of the transient response when acetate formation starts. The predicted effect was verified in fed-batch cultivations of E. coli TOPP1 and E. coli BL21(DE3), both before and after induction of recombinant protein production. It was also observed that the critical specific glucose uptake rate, at which acetate formation starts, was significantly decreased after induction. On-line detection of acetate formation with a standard sensor opens up new possibilities for feedback control of substrate feeding