Claus Lindvald Johansen

On-line estimation of biomass, glucose and ethanol in Saccharomyces cerevisiae cultivations using in-situ multi-wavelength fluorescence and software sensors

Batch bioreactor cultivations using Saccharomyces cerevisiae at high (190-305 gl(-1) glucose) or low (21-25 gl(-1) glucose) gravity conditions were monitored on-line using multi-wavelength fluorescence (MWF) and standard monitoring sensors. Partial least squares models were calibrated for the prediction of cell dry weight (CDW), ethanol and consumed glucose, using the two data types separately. The low gravity cultivations (LGCs) consisted of two phases (glucose consumption with concomitant ethanol production followed by ethanol consumption after glucose depletion), which proved difficult to model using one and the same model for both phases. Segmented modelling, using different models for the two phases, improved the predictions significantly. The prediction models calibrated on standard on-line process data displayed similar or lower root mean square error of prediction (RMSEP) compared to the fluorescence models. The best prediction models for high gravity cultivations (HGCs) had RMSEPs of 1.0 gl(-1) CDW, 1.8 gl(-1) ethanol and 5.0 gl(-1) consumed glucose, corresponding to 4%, 2% and 2% of the respective concentration intervals. Corresponding numbers in low gravity models were 0.3 gl(-1) CDW, 0.7 gl(-1) ethanol and 1.0 gl(-1) consumed glucose, corresponding to 4%, 8% and 4% of the respective concentration intervals.

Influence of morphology on product formation in aspergillus awamori during submerged fermentations

The relationship between fungal morphology and heterologous protein production was examined for an Aspergillus awamori strain during a series of fermentations with a batch phase followed by a fed‐batch phase. Agitation rate and inoculation concentration were used as controlled variables to generate different fungal morphologies in 20‐dm3 stirred tank reactors. Morphology was quantitatively characterized using Image Analysis. The different agitation rates and inoculum concentrations had large effects on the development in hyphal length and number of tips during the fermentations. A reduced inoculum concentration resulted in a more branched mycelium. The different agitation rates affected the morphology after 30 h of fermentation significantly but did not affect the start time of fragmentation. A 3‐fold increase in hyphal length increased the apparent viscosity by a factor of 7. The observed morphological differences had only a limited effect on product formation, suggesting that the structural features such as hyphal length and number of tips are of less importance for product formation. The primary effect of morphology on product formation is due to viscosity.

Culture fluorescence measurements during batch and fed-batch cultivations with Penicillium chrysogenum

Abstract A commercially available sensor from Ingold AG has been used to measure the NAD(P)H-dependent culture fluorescence and the signal has been analyzed, especially with respect to possible interferences from components present in complex media used during penicillin cultivations. Penicillin V interfers strongly with the measurement, and this makes it difficult to use the sensor for control of fed-batch penicillin cultivations. For growth on defined media the fluorescence signal does, however, contain valuable information concerning the redox state of the cells, e.g., the formation and subsequent consumption of organic acids such as gluconic acid and lactic acid. Thus, during the metabolism of gluconic acid the NAD(P)H level in the cells is high, and this strongly indicates that this compound is canalized through the pentose phosphate pathway during its metabolism. Apart from its use in physiological studies the sensor is attractive for evaluation of the initial phase of fed-batch penicillin cultivations and the fluorescence signal can be used to specify the optimal time to switch from batch to fed-batch operation.

Simulation of penicillin production in fed-batch cultivations using a morphologically structured model

A mathematical model is formulated to describe trends in biomass and penicillin formation as well as substrate consumption for fed-batch cultivations. The biomass is structured into three morphological compartments, and glucose and corn steep liquor are considered as substrates for growth. Penicillin formation is assumed to take place in the subapical compartment and in the growing region of the hyphal compartment. Furthermore, it is inhibited by glucose. Model parameters are estimated using an evolutionary algorithm and fitting the model to a standard fed-batch cultivation. The model is validated on experimental data from three different fed-batch cultivations, including two repeated fed-batch cultivations. The model predictions show good agreement with the measurements of biomass and pencillin concentrations for all fed-batch cultivations. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 593-604, 1997.

On-line monitoring of penicillin V during penicillin fermentations : a comparison of two different methods based on flow-injection analysis

Abstract Two different flow-injection analysis (FIA) methods used for on-line monitoring of penicillin V during fed-batch penicillin fermentations are compared. The fermentations are carried out using a high yielding strain of Penicillium chrysogenum and both analyzers are used continuously for on-line monitoring during 200 h. Both methods are based on a reaction catalyzed by β-lactamase, which converts penicillin V to penicilloic acid. In analyzer 1 the sample is injected into a buffer stream and carried to a stirred measurement cell containing an enzyme electrode. The enzyme electrode consists of β-lactamase immobilized onto a sensitive pH glass electrode with a very low response time. No predilution of the sample is required in this analyzer. In analyzer 2 the sample is injected into an enzyme reactor containing immobilized β-lactamase. The formed penicilloic acid is detected by decolorization of an iodine-starch complex. By means of a second injection valve it is possible to have the sample by-pass the enzyme reactor and instead pass a dummy reactor in which no β-lactamase is immobilized. Thereby both the concentration of penicillin and penicilloic acid can be measured. On-line dilution of the sample is ensured by a gradient technique. Experimental results from on-line monitoring of penicillin V using the two methods are presented. The large amount of precise measurements enables a very good resolution of the penicillin production; something of extreme value for verification of mathematical models describing the fermentation kinetics.

Monitoring and control of fed-batch penicillin fermentation

Abstract An automized system for monitoring and control of fed-batch penicillin fermentations on complex media is described. The system consists of three highly automated bioreactors equipped with on-line sensors and analytical systems for on-line monitoring of culture fluorescence, exhaust gas composition and ammonia. All data are collected on a process computer containing a 68000 Motorola processor. The computer is programmed in PEARL using RTOS as operating system, a combination which allows multitasking programming.