We assess the effect of substrate heterogeneity on the metabolic response of P. chrysogenum in industrial bioreactors via the coupling of a 9-pool metabolic model with Euler-Lagrange CFD simulations. In this work, we outline how this coupled hydrodynamic-metabolic modeling can be utilized in 5 steps. (1) A model response study with a fixed spatial extra-cellular glucose concentration gradient, which reveals a drop in penicillin production rate qp of 18–50% for the simulated reactor, depending on model setup. (2) CFD-based scale-down design, where we design a 1-vessel scale down simulator based on the organism lifelines. (3) Scale-down verification, numerically comparing the model response in the proposed scale-down simulator with large-scale CFD response. (4) Reactor design optimization, reducing the drop in penicillin production by a change of feed location. (5) Long-term fed-batch simulation, where we verify model predictions against experimental data, and discuss population heterogeneity. Overall, these steps present a coupled hydrodynamic-metabolic approach towards bioreactor evaluation, scale-down and optimization.@en

In a 54 m3 large-scale penicillin fermentor, the cells experience substrate gradient cycles at the timescales of global mixing time about 20–40 s. Here, we used an intermittent feeding regime (IFR) and a two-compartment reactor (TCR) to mimic these substrate gradients at laboratory-scale continuous cultures. The IFR was applied to simulate substrate dynamics experienced by the cells at full scale at timescales of tens of seconds to minutes (30 s, 3 min and 6 min), while the TCR was designed to simulate substrate gradients at an applied mean residence time ((Formula presented.)) of 6 min. A biological systems analysis of the response of an industrial high-yielding P. chrysogenum strain has been performed in these continuous cultures. Compared to an undisturbed continuous feeding regime in a single reactor, the penicillin productivity (qPenG) was reduced in all scale-down simulators. The dynamic metabolomics data indicated that in the IFRs, the cells accumulated high levels of the central metabolites during the feast phase to actively cope with external substrate deprivation during the famine phase. In contrast, in the TCR system, the storage pool (e.g. mannitol and arabitol) constituted a large contribution of carbon supply in the non-feed compartment. Further, transcript analysis revealed that all scale-down simulators gave different expression levels of the glucose/hexose transporter genes and the penicillin gene clusters. The results showed that qPenG did not correlate well with exposure to the substrate regimes (excess, limitation and starvation), but there was a clear inverse relation between qPenG and the intracellular glucose level.@en

The compartment model (CM) is a well-known approach for computationally affordable, spatially resolved hydrodynamic modeling of unit operations. Recent implementations use flow profiles based on Computational Fluid Dynamics (CFD) simulations, and several authors included microbial kinetics to simulate gradients in bioreactors. However, these studies relied on black-box kinetics that do not account for intracellular changes and cell population dynamics in response to heterogeneous environments. In this paper, we report the implementation of a Lagrangian reaction model, where the microbial phase is tracked as a set of biomass-parcels, each linked with an intracellular composition vector and a structured reaction model describing their intracellular response to extracellular variations. A stochastic parcel tracking approach is adopted, in contrast to the resolved trajectories used in CFD implementations. A penicillin production process is used as a case study. We show good performance of the model compared with full CFD simulations, both regarding the extracellular gradients and intracellular pool response, using the mixing time as a matching criterion and taking into account that the mixing time is sensitive to the number of compartments. The sensitivity of the model output towards some of the inputs is explored. The coarsest representative CM requires a few minutes to solve 80 h of flow time, compared with approximately 2 weeks for a full Euler–Lagrange CFD simulation of the same case. This alleviates one of the major bottlenecks for the application of such CFD simulations towards the analysis and optimization of industrial fermentation processes.@en

In large-scale bioreactors, there is often insufficient mixing and as a consequence, cells experience uneven substrate and oxygen levels that influence product formation. In this study, the influence of dissolved oxygen (DO) gradients on the primary and secondary metabolism of a high producing industrial strain of Penicillium chrysogenum was investigated. Within a wide range of DO concentrations, obtained under chemostat conditions, we observed different responses from P. chrysogenum: (i) no influence on growth or penicillin production (>0.025 mmol L−1); (ii) reduced penicillin production, but no growth limitation (0.013–0.025 mmol L−1); and (iii) growth and penicillin production limitations (<0.013 mmol L−1). In addition, scale down experiments were performed by oscillating the DO concentration in the bioreactor. We found that during DO oscillation, the penicillin production rate decreased below the value observed when a constant DO equal to the average oscillating DO value was used. To understand and predict the influence of oxygen levels on primary metabolism and penicillin production, we developed a black box model that was linked to a detailed kinetic model of the penicillin pathway. The model simulations represented the experimental data during the step experiments; however, during the oscillation experiments the predictions deviated, indicating the involvement of the central metabolism in penicillin production.@en

The objective of this research study is to be able to scale-up and optimize aerobic fermentation processes via computer simulations. As an example the production of penicillin by P. chrysogenum is studied. In vivo kinetics of the cell can be understood by conducting stimulus response and scale down experiments. These experiments provide dynamic information that can be used to estimate parameters of enzyme kinetic models. The transport phenomena inside the bioreactor can by understood by solving hydrodynamic model using computational fluid dynamics (CFD). Thus, with information from intracellular and extracellular environments, an integrated model is solved using CFD. In these simulations, the multiphase flow and transport phenomena taking place inside the bioreactor are simulated, and integrated with the microbial reaction kinetics. The micro-organisms inside the reactor are followed along their trajectories, where they experience a changing environment. The dynamic response of the cells to this environment dictates the productivity and selectivity of the cells. By simulating both the flow in the reactor and the response of the cells, the entire process inside the bioreactor can be assessed and scaled-up and optimization can be done without the need of costly experiments, both in time and financial resources.@en

The objective of this research study is to be able to scale-up and optimize aerobic fermentation processes via computer simulations. As an example the production of penicillin by P. chrysogenum is studied. In vivo kinetics of the cell can be understood by conducting stimulus response and scale down experiments. These experiments provide dynamic information that can be used to estimate parameters of enzyme kinetic models. The transport phenomena inside the bioreactor can by understood by solving hydrodynamic model using computational fluid dynamics (CFD). Thus, with information from intracellular and extracellular environments, an integrated model is solved using CFD. In these simulations, the multiphase flow and transport phenomena taking place inside the bioreactor are simulated, and integrated with the microbial reaction kinetics. The micro-organisms inside the reactor are followed along their trajectories, where they experience a changing environment. The dynamic response of the cells to this environment dictates the productivity and selectivity of the cells. By simulating both the flow in the reactor and the response of the cells, the entire process inside the bioreactor can be assessed and scaled-up and optimization can be done without the need of costly experiments, both in time and financial resources.@en

The objective of this research study is to be able to scale-up and optimize aerobic fermentation processes via computer simulations. As an example the production of penicillin by P. chrysogenum is studied. In vivo kinetics of the cell can be understood by conducting stimulus response and scale down experiments. These experiments provide dynamic information that can be used to estimate parameters of enzyme kinetic models. The transport phenomena inside the bioreactor can by understood by solving hydrodynamic model using computational fluid dynamics (CFD). Thus, with information from intracellular and extracellular environments, an integrated model is solved using CFD. In these simulations, the multiphase flow and transport phenomena taking place inside the bioreactor are simulated, and integrated with the microbial reaction kinetics. The micro-organisms inside the reactor are followed along their trajectories, where they experience a changing environment. The dynamic response of the cells to this environment dictates the productivity and selectivity of the cells. By simulating both the flow in the reactor and the response of the cells, the entire process inside the bioreactor can be assessed and scaled-up and optimization can be done without the need of costly experiments, both in time and financial resources.@en