G.C. Paul

Dependence of mycelial morphology on impeller type and agitation intensity.

The influence of the agitation conditions on the morphology of Penicillium chrysogenum (freely dispersed and aggregated forms) was examined using radial (Rushton turbines and paddles), axial (pitched blades, propeller, and Prochem Maxflow T), and counterflow impellers (Intermig). Culture broth was taken from a continuous fermentation at steady state and was agitated for 30 min in an ungassed vessel of 1.4‐L working volume. The power inputs per unit volume of liquid in the tank, P/VL, ranged from 0.6 to 6 kW/m3. Image analysis was used to measure mycelial morphology. To characterize the intensity of the damage caused by different impellers, the mean total hyphal length (freely dispersed form) and the mean projected area (all dispersed types, i.e., also including aggregates) were used. [In this study, breakage of aggregates was taken into account quantitatively for the first time.]

A structured model for hyphal differentiation and penicillin production using Penicillium chrysogenum

A structured kinetic model describing growth, differentiation, and penicillin production in submerged Penicillium chrysogenum fermentations is reported. The filamentous hyphae are divided into four distinct regions on the basis of the activities and structure of hyphal compartments, viz., actively growing (mainly apical) regions, nongrowing or penicillin producing regions, vacuoles, and degenerated or metabolically inactive regions. A mechanistic approach is taken to give quantitative descriptions of differentiation and degeneration as a consequence of vacuolation. The growth and degeneration of vacuoles are expressed in the form of a population balance. The model assumes that newly generated vacuoles appear by differentiation of healthy regions, grow in size with limitation of available substrate, and eventually give rise to empty hyphal compartments. In the model the penicillin production is related to the amounts of the nongrowing regions of the hyphae. The model is used for successful predictions of the amounts of the four hyphal regions and the penicillin G production rate throughout the fed‐batch fermentations of an industrial P. chrysogenum strain under different glucose feeding regimes. Quantitative information on proportions of the hyphal regions was obtained from image analysis measurements and the parameters of the kinetic model were identified. When the glucose feed rate to the production culture is switched between a high and a low value, the model can successfully predict the dynamic changes of differentiation and the resulting penicillin production caused by the variations in the nutrient conditions. The use of image analysis to characterize differentiation as a basis for structured modeling of the penicillin fermentation appears to be very powerful, and the method has great potential for use in process simulation and control of antibiotic fermentations.

Effect of biomass concentration and mycelial morphology on fermentation broth rheology.

The effect of biomass concentration and mycelial morphology on fungal fermentation broth rheological properties has been investigated. In previous work it had been shown that commonly used rheological parameters, such as the power law consistency and flow behavior indices, could be correlated successfully with the broth biomass concentration and clump morphological parameters of roughness and compactness. More recent work on a broader range of data showed a correlation between roughness and compactness; consequently, it was not correct to use both of these morphological variables simultaneously in rheological parameter correlations. Furthermore, earlier correlations were only made using clump morphological parameters, as clumps were found to be around 90% of the biomass in batch fermentations. In the present work it was found that the percentage of clumps fell to around 30% to 40% of a sample during the later stages of fed-batch fermentations. No clear relationship between the flow behavior index and biomass concentration was found, at least for those phases of the fermentation in which the viscosities were high enough for the rheology to be characterized by a disk turbine rheometer. The mean value of the flow behavior index was found to be 0.35 +/- 0.1 (standard deviation) throughout both batch and fed-batch fermentations, although some significant deviations from this value were observed early and very late in the fermentations. Correlations for the consistency index, measured using a disk turbine rheometer, were based on the biomass concentration and the mycelial size (represented by the mean projected area or the mean maximum dimension of all the mycelia). These correlations were reasonably successful for both fed-batch and batch fermentations. The correlation using the mean maximum dimension was preferred to that using the mean projected area, as the former is independent of magnification. The proposed correlation is: where K is the consistency index (Pa. s(n>)), C(m) is the biomass concentration as dry cell weight (g L(-1)), and D is the mean maximum dimension (microm). It should be noted that small changes in the exponent on the biomass concentration (alpha) may dramatically affect any predictions. Consequently, caution in the use of this correlation (and that based on mean projected area) is advocated, although its accuracy may be suitable for operational or design purposes.

Agitation induced mycelial fragmentation of Aspergillus oryzae and Penicillium chrysogenum.

Given the impact of mycelial morphology on fermentation performance, it is important to understand the factors that influence it, including agitation-induced fragmentation. The successful application of the energy dissipation/circulation function (EDC) to correlate fragmentation of Penicillium chrysogenum with agitation intensity and with different impeller types [5] has already been demonstrated. The EDC function takes into account the specific energy dissipation rate in the impeller swept volume and the frequency of mycelial circulation through that volume. In order to explore whether the EDC function can be used more generally to correlate fragmentation of different filamentous species, the present study extended the concept to agitation-induced, off-line fragmentation of Aspergillus oryzae grown in chemostat culture. The work shows that at EDC values off-line greater than that in the chemostat, fragmentation with different impellers can be correlated with the EDC. For EDC values less than those used in the chemostat, fragmentation did not occur. The earlier results of Jüsten et al. [5] with Penicillium chrysogenum are also reconsidered and found to behave similarly.

A structured model for penicillin production on mixed substrates

Abstract A structured kinetic model previously developed to describe the growth, differentiation, and penicillin production of Penicillium chrysogenum has been enhanced and extended in order to apply it to a mixed carbon source fermentation. The filamentous hyphae are divided into four distinct regions on the basis of their activities and the physiological structure (i.e., vacuolation) of the hyphal compartments: viz., actively growing (mainly apical) regions, non-growing or penicillin producing regions, vacuoles, and degenerated or metabolically inactive regions. A simple approach is taken to give quantitative descriptions of hyphal extension, branch formation, vacuolation and differentiation. The fermentation medium contained glucose and lactose monohydrate as the main carbon sources. The source of the lactose was whey powder used in excess in the inoculum medium, whilst glucose was fed continuously throughout the fermentation. Lactose, a disaccharide, is hydrolysed to two monosaccharides, glucose and galactose, when the residual glucose concentration in the medium drops to a very low level. The utilisation of glucose and that of galactose following the hydrolysis of lactose were observed to occur simultaneously. This allowed the assumption of simple lactose utilisation kinetics in which lactose hydrolysis could be considered as producing an equivalent amount of glucose. The model has been used for successful predictions of fed-batch penicillin fermentations using an industrial P. chrysogenum strain under different glucose feed rates. Quantitative information on proportions of the hyphal regions was obtained from image analysis measurements and the parameters of the model were identified. When the glucose feed rate to the production culture was switched between a high and a low value, the model successfully predicted the dynamic changes of differentiation and the resulting penicillin production caused by the variations in the nutrient conditions. The use of image analysis to characterise differentiation as a basis for structured modelling of the penicillin fermentation appears to be very powerful, and such models have great potential for use in process simulation and control of antibiotic fermentations.

Towards intelligent process supervision: Industrial penicillin fermentation case study

Abstract Due to the their biological nature and inherent variability bioprocesses place significant demands on even the most advanced supervision approaches. Sophisticated supervision software, such as G2 from Gensym (a real-time knowledge based system) is already finding wide industrial bioprocess application. However, in order to overcome the severe challenges posed a straightforward ‘coding’ of operators and engineers knowledge is not sufficient. What is required is a methodology which attempts to maximise the information available for supervision purposes. This paper describes the development of an application in which feature extraction and data based methodologies are integrated with sophisticated physiological models to considerably enhance a rule-based supervisory system. Together within the G2 real-time knowledge based system framework they offer the potential for bioprocess operational improvement.

Hybrid modelling for on-line penicillin fermentation optimisation

Abstract This paper describes the procedures that are necessary to arrive at a model that is sufficiently accurate to be used in an on-line penicillin fermentation optimisation scheme. A structured mechanistic model developed previously was available but this model failed to account for the effects of low levels of dissolved oxygen on growth and production. When moving towards optimising the fermentation, dissolved oxygen becomes the parameter limiting process productivity and hence it is important to be able to account for its process influence. Terms predicting dissolved oxygen changes and describing its effect when at low levels were included in the model to compensate for the reduction in growth and production. Even so, the natural variation experienced in the fermentation resulted in process / model mismatch. On line correction of model coefficients via an observer approach provided the accuracy required for optimisation purposes. The improvements in the accuracy of the model predictions are demonstrated.

Improving the Estimation of Parameters of Penicillin Fermentation Models

Abstract Models for use in control and estimation applications should match the process as closely as possible. Fermentation process models are usually complex, containing many states and parameters. Obtaining accurate estimates of the parameters of such models is a costly and time-consuming process. Here we show a way of reducing the time and cost by designing optimal experiments for parameter identification. The method presented uses genetic algorithms to search for input profiles which optimise scalar functions of the Fisher information matrix, thus maximising the improvement in the parameter estimates that may be obtained from each experiment performed. The Penicillium chrysogenum penicillin-G fermentation, a secondary metabolite fermentation, is used as an example.

Image Analysis for Quantification of Fungal Biomass and Differentiation

Abstract Fully-automatic image analysis methods are described for the measurement of vacuolation in filamentous fungi growing in dispersed form in submerged cultures. For Penicillium chrysogenum biomass from 0.03 to 38 gL -L , volumes of cytoplasmic and vacuolised or degenerated regions can be converted successfully into dry weight estimations, even when the medium contains suspended solids up to 30 gL -L . The vacuolation can be characterised in detail for physiological studies. For example, size and shape distributions of vacuoles can be measured, and are presented across the time course of a test fed-batch penicillin fermentation. Although off-line, the method is rapid enough (30 min per sample) to be considered for use as a process control tool. Links between cytoplasmic content and antibiotic production are being sought. The method might be used to provide data for structured models of fungal fermentations, and for control of product formation in those fermentations.

Measurement of Cellular Differentiation of Filamentous Fungi using Image Analysis

Abstract An image analysis method has been developed to measure cellular differentiation of filamentous fungi growing in dispersed form in submerged culture. The proportions of vacuoles and empty cells, and growing (mainly apical) and non-growing regions in the mycelia have been quantified. The shape and size distributions of vacuoles and empty cells in the hyphae were also obtained. It has been demonstrated with Penicillium chrysogenum that these differentiation states might be manipulated by the nutrient conditions in the fermentation. The method permits rapid and accurate quantitative studies of fungal physiology and might be used for the improvement of process models and process control strategies for antibiotic fermentations.