The growing global population and heightened concern for climate change
leads to increased interest in utilizing microbial fermentations to replace polluting production processes for e.g., plastics, fuels, and animal proteins. Computational fluid dynamics (CFD) is a valuable tool for accelerating the scale-up and optimization of large scale bioprocesses. However, the design correlations underlying most CFD models are validated with air-water systems, not accounting for the distinct hydrodynamic properties of microbial fermentation broth. This gives rise to the first research question: What solutes affect bubble column bioprocess performance the most? In chapter 2, we provide an extensive overview of the current understanding of how various biotechnologically relevant solutes impact the hydrodynamics of bubble columns. We examine the effects of components found in fermentation broths, including salts, surfactants, viscoelastic solutes, alcohols, acids, ketones, sugars, biomass, and proteins, on mass transfer, bubble formation, bubble interactions,
and flow regime transitions. These components all exhibit unique effects
which are coarsely understood for some, yet their combined influences remain poorly characterized. Characterization of the concentration at which coalescence inhibition occurs for different compounds, especially in mixtures,
is identified as an important avenue for future research.

Gas–liquid mass transfer is a key performance metric in bioprocess engineering, yet the impact of electrolytes in the medium remains poorly understood.
Chapter 3 investigates the influence of biotechnologically relevant salts on
bubble coalescence, gas fraction, and interfacial area in a 30 L bubble column.
This chapter investigates the second research question: Can the Gibbs-Marangoni pressure be used to predict the effects of all electrolytes on bubble coalescence behavior across scales? Fiber optic probes and pressure probe measurements quantify the effects of salt concentration and ionic strength on bubble size distribution (BSD) and gas fraction. The shortcomings of both concentration and ionic strength for a unified prediction of BSD and gas fraction
are demonstrated and overcome by calculating the Gibbs-Marangoni pressure (p_GM ) resulting from the salt concentration. The p_GM is the force that hinders film drainage between bubbles, and we find that it describes the effects of diverse electrolytes across different experimental setups. Our findings reveal a path to improved reactor design and optimization of gas-liquid mass transfer in fermentations and other bioprocesses.

High-resolution experimental data on local gas fraction and bubble size distribution are essential for reducing the uncertainties in bubble column performance predictions. Unfortunately, experimental characterization of gas-phase dynamics at appreciable gas flow rates and gas fractions is challenging in
clear fluids and fermentation broths alike. Chapter 4 investigates a 3-angled
X-ray tomography setup for measurement of spatially resolved gas fraction
profiles in a 20 cm internal diameter bubble column with 100 cm static liquid
height. This chapter investigates the third research question: How can a 3- angled tomography setup be used to obtain accurate radial gas fraction profiles
in bubble columns with an inner diameter larger than 15 cm?

X-ray scatter and beam hardening are identified as main sources of measurement error, and corrections for these phenomena are proposed and tested. It was found that an increase in X-ray tube voltage from 120 kV through 135 kV to 150 kV is not enough to mitigate beam hardening effects. Software-based post-processing image corrections were found satisfactory at mitigating the effects of both beam hardening and cross-scatter. Comparison of tomographic reconstructions of the local gas fractions in our experiments with relevant literature values showed that significant post-processing is required to produce trustworthy holdup profiles. Reconstruction with beam hardening correction or combined beam hardening and cross-scatter correction produced profiles closely matching literature data at superficial gas velocities of 1.7 cm s⁻¹, 4.6 cm s⁻¹, 5.8 cm s⁻¹ and 8.6 cm s⁻¹. Comparison between X-ray tomography
and radial profiles from optical fiber probe measurements in the same column
showed a discrepancy, with the fiber probe reporting roughly 20 %lower gas
fraction values. Pressure probe measurements of the mean holdup agreed
with the fiber probe results. An extra correction term might be needed to
bring X-ray tomography into quantitative agreement with the complementary measurement methods.

Overall, chapter 4 shows that three-angled X-ray tomography allows for rapid
and reliable acquisition of gas fraction profiles in scale-invariant bubble columns. The method is a factor twelve faster than optical fiber probes, with increased radial resolution, but lacking the spatial and temporal resolution to identify single bubbles. The combination of X-ray tomography for gas fraction and fiber probes for bubble size distribution measurement is an attractive setup
for systematic studies of the effects of superficial gas velocity and fluid composition on gas distribution and interfacial area in bubble columns.
Future work with the X-ray tomography (XRT) setup can move towards characterization of the temporal dynamics in bubble columns with biotechnologically relevant liquids. Furthermore, the characterization of actual fermentation broth can serve as a challenge to see if the p_GM framework can provide meaningful descriptive power in real-world scenarios. ... Read more

Bubble column reactors (BCRs) are valuable in the biochemical industry for their efficient gas-liquid mass transfer and mixing capabilities. A key performance parameter in BCRs is gas holdup, which can be significantly influenced by components present in fermentation broths. This study investigated the effect of the important fermentation products ethanol, n-propanol, iso-propanol, n-butanol, acetic acid, and lactic acid on gas holdup. These components impact bubble coalescence via the so-called Marangoni effect. This study correlates gas holdup with the dimensionless Marangoni number (Ma) in BCRs. The model proposed by Wang et al. [1] successfully predicts 87% of the measured holdup values within a 10% error margin.
Additionally, correlations for gas holdup distribution are compared, with Schweitzer et al. [2] providing the most accurate local predictions (70% within 10% error). This study provides a comparative overview of gas holdup correlations relevant to BCRs and demonstrates the significance of incorporating the Marangoni effect in predictive models.
Research on local gas holdup in BCRs often requires invasive measurement techniques. X-ray tomography (XRT) enables measurements of gas without disruption of the flow associated with traditional methods such as optical fiber probes. However, noise from X-ray scattering and artifacts from beam hardening still hinder accurate quantification of gas holdup. This study presents a methodology for processing X-ray data to overcome these challenges. X-ray data corrected for scatter and beam hardening results in tomographic reconstructions with a gas holdup profile similar to what was found with an optical fiber probe. This correction methodology can assist other researchers using similar X-ray setups by improving the accuracy of gas holdup measurements in multiphase systems. ... Read more

As the world increasingly decarbonises, there is an increasing pressure on chemical manufacturing to move away from fossil carbon sources. Industrial bioprocesses provide one such alternative for fossil carbon. Bubble columns as a bioreactor type are particularly well suited to such large scale applications. However, models of bubble columns, whether based on
design correlations or computational fluid dynamics, have been shown to break down when
air-water systems are replaced with systems containing actual fermentation broth. The additional broth components can significantly affect interphase mass transfer through e.g. limiting bubble breakup, which can in turn make or break the economics of a bioprocess [1]. Experimental data on the effects of broth components on the physics in bubble columns is essential to develop better models. Gathering such data requires experimental methods capable of penetrating the industrially-relevant but opaque churn-turbulent flows. A promising method for determining the state of a large section of the bubble field in a bubble column is X-ray computed tomography. The TU Delft X-ray tomography setup seeks to achieve this using three source-detector pairs capable of capturing X-ray data at high frame rates. However, in order to be able to apply any experimental technique, it must first be validated and the sources of and magnitude of its various measurement errors must be quantified.
This thesis uses computational fluid dynamics to validate tomographic reconstruction algorithms.
The computational fluid dynamics model was validated using experimental data from
Sanyal et al.[2] Furthermore, this thesis finds ways of improving tomographic reconstructions
through discovering which reconstruction algorithms perform best for different datasets. It was found that for time-resolved bubble fields, a version of SIRT (Simultaneous Iterative Reconstruction Technique) with generalised Tikhonov regularisation using the derivative operator performed best with a NRMSE (Normalised Root Mean Squared Error) of 0.0867 over a baseline value of 0.1123 using the default SIRT method and an F-score of 0.641 for the binary
classification of air and water. For time-averaged reconstructions of the gas holdup, an SIRT
with standard Tikhonov regularisation with an offset to the mean gas holdup was found to
perform best with a NRSME of 0.0137 over a SIRT baseline of 0.0160. Finally, this thesis
shows the improvement to tomographic reconstructions for an upgraded version of the TU
Delft X-ray tomography setup and provides recommendations for future research on this topic.
It was shown that increasing the number of source-detector pairs to five, leads to significant
improvements in the time-resolved bubble field reconstructions, with a new NRMSE of 0.0617
(-28%) and F-score of 0.823 (+28%) ... Read more

One of the major challenges mankind faces nowadays is combating climate change. A substantial fraction of greenhouse gases are released by industrial processes, as steelmaking, (oil)refinery and waste processing. Emissions from these processes can partly be prevented with a recently developed technology called gas fermentation. Within this process, synthesis gas – amixture containing CO, CO2 and H2 – is converted into ethanol and acetic acid by bacteria such as Clostridium autoethanogenum. These products could be used in a wide range of applications, like fuels, plastics and cosmetics. Whilst gas fermentation is already applied at commercial-scale, challenges in scale-up persists due to complex multi-scale interactions among the bioreactor, gas bubbles, and bacteria. The poor solubility of CO and H2 alongside gas bubble coalescence, leads to low gas-to-liquid mass transfer rates (typically denoted via kLa). Slow mixing in industrial bioreactors (500m3), and high gas conversion rates, result in large spatial variations in dissolved gas concentrations. Bacteria experiencing concentration fluctuations have often been related to decreased process performance... ... Read more

Presented works describe a novel approach to assess mixing in a stirred vessel using light-based tomography. The study is driven by key research questions: What are the obstacles in three-dimensional dynamic tracer distribution reconstruction? How should the experimental equipment be constructed to obtain the best possible data from three cameras recording the back-lit stirred tank? How can raw images be processed to isolate the ray-dye interaction? And, how can relevant mixing information be obtained from projections and reconstructed volumetric data? The research begins with a short introduction of traditional mixing measurement techniques, establishing the context and relevance of the work. The theoretical background of the study is then presented, including the principles of tomographic reconstruction and the main algorithms used in the process. The methodology involves the use of synthetic data obtained from LES for the framework and baseline creation followed by the acquisition of experimental data. A significant part of the methodology is dedicated to image pre-processing, which incorporates as main steps the inverted grayscale transformation, brightness normalization, background removal using image similarity metrics and object removal with the use of a neural network. The use of the simplistic forward model based on the Lambert-Beer law is described, followed by the implementation of the projection matrix-free Simultaneous Algebraic Reconstruction Technique. The outcomes of both synthetic and experimental data reconstruction are presented and despite the shortcomings of the used experimental setup the 2D and 3D mixing maps were created, supported by the local Coefficient of Variance calculation to gain further insight into the process. The conclusions highlight the potential of light-based tomography for evaluating mixing while acknowledging the need for significant refinement and validation of the methodology. Recommendations include the improvement of imaging, equipment modifications, and reconstruction implementation. ii ... Read more

In recent years, biotechnological processes have gained increased interest due to their potential for high-value compound production and waste recycling. This shift towards biotechnology is driven by global challenges such as food security, climate change, and the transition to renewable resources. To address the limitations of large-scale fermentations, scale-down approaches have been recommended to minimize microbial performance losses during scale-up procedures. Computational fluid dynamics (CFD) coupled with omics-based technologies offer valuable insights into the environmental and intracellular
lifelines of cells. However, current laboratory-scale setups have certain limitations, emphasizing the need for dynamic microfluidic single-cell cultivation (dMSCC) devices. These devices enable the analysis of single-cell behavior in dynamic environments with high temporal resolution.

This thesis focuses on improving the amplitude control while maintaining temporal resolution in dMSCC devices. A new dMSCC device design was analyzed using a 2D model, which was experimentally validated. The results demonstrated that the design mechanism effectively generated concentration profiles resembling discrete and smooth lifelines, albeit with a relatively high response time (30 seconds). A mesh independence study indicated minimal deviations (2 %) in results for different mesh refinements, while complex geometric structures introduced greater variations.

The experimental validation of the 2D COMSOL Multiphysics model highlighted discrepancies between the experimental data and model predictions, both at the outlets of the microfluidic concentration gradient generator (μCGG) and inside the chamber (RMSE=0.1-0.75; >10% of experimental data). However,
the observed trends of the concentration profiles inside the chamber were well-captured. Optimization studies were conducted based on these findings, leading to valuable conclusions. Narrowing the chamber width increased the chip’s response time. Moreover, increasing the space between μCGG outlets
as well as increasing fluid velocity inside the μCGG (while keeping the maximum velocity constant) improved gradient width. The latter approach is preferred to maintain temporal resolution. A comparison between COMSOL Multiphysics (RMSE=0.14) and Ansys Fluent (RMSE=0.15) models revealed that Ansys Fluent better captures experimental trends but has lower prediction accuracy. Further investigations involved a Design of Experiments (DoE), which indicated that the current μCGG design is suitable for fluid velocities preferably lower than 1 · 10−5 m/s and tracers with high diffusion coefficients. These conclusions provide insights into optimizing dMSCC devices and contribute to the broader understanding of mimicking microbial lifelines. ... Read more

Fermentation processes are considered to be essential to decrease our reliance on fossil fuel based products. However, the scale-up from lab-scale to industrial-scale has proven to be difficult. Computational Fluid Dynamics (CFD) has the potential to be a tool to optimize the scale-up and to help engineers understand the relevant hydrodynamics inside such reactors. However, traditional CFD simulations are computationally intensive and it is not uncommon that simulations can take several months to compute a few minutes of flow-time. Due to the recent development of GPU-based hardware, the Lattice-Boltzmann method (LBM) has been gaining much interest as this meant an orders-of-magnitude decrease of needed computational time compared to conventional CFD methods. In order to calibrate the models underlying the simulations, the obtained results of said simulations should be validated against experimentally obtained data, which is exceptionally scarce for industrial-scaled reactors. Hence the aim of this thesis is to to investigate the applicability of the LBM in high gas-flow, industrial sized reactors by studying three benchmark cases. Using Large Eddy Simulations (LES) and Euler-Lagrangian tracking of bubble parcels, the models provided by M-Star (MStar Simulations, LLC) are validated by replicating a small-scale reactor of which the LBM has already been successfully applied to. The industrial-scaled case consists of the simulation of the 22m3 industrial reactor located in Stavanger, which is an exception regarding the scarcity of experimental data for industrial sized reactors. As this data set did not encompass all the relevant data for aerated stirred tanks, the suitability of the LBM and provided models are also tested for a smaller scaled tank of which the Bubble Size Distribution (BSD) throughout the vessel is known. The applicability of the provided models will be tested by comparing the obtained gas hold-up, BSD and power consumption with experimental data sets. Although the LBM is suitable for industrial-scaled reactors, the provided models by M-Star are not sufficient to predict the gas hold-up and power consumption at high superficial gas velocities. The provided Free Particle drag correlation is not sufficient to describe the dispersion of bubbles throughout the vessel, leading to a significantly under-estimated gas hold-up. In addition, the parcel approach leads to the formation of large bubbles in the vessel. And although enforcing a maximum coalescence diameter does improve the BSD, the gas hold-up is not significantly influenced by the presence of large bubbles. Furthermore, the Euler-Lagrangian way of modeling bubble particles did not lead to the formation of gas cavities, which resulted in an insignificant drop in power consumption. Nevertheless, the current work provides a foundation for subsequent research. ... Read more

The traditional Eulerian view of biomass in bioprocess modelling results in issues when modelling large-scale bioreactors in which heterogeneous conditions are common. As the field increasingly moves from “scale-up” to “scale-down” philosophy, in which such heterogeneities are included from the start of process design, accurate modelling of the biomass response to these varying conditions is essential. Euler-Lagrange (EL) simulations provide a means of modelling the microbial lifelines of cells traversing heterogenous conditions of a bioreactor. Lapin et al. were the pioneers of EL simulation in their 2004 paper where a metabolic model of glycolysis is coupled to a Lagrangian biomass phase. This BSc thesis focusses on reproducing their model using modern computational fluid dynamics (CFD) techniques. Specifically, by using a dynamic Lattice Boltzmann Method using Large Eddy Simulation model for CFD as opposed to a frozen-flow Finite Volume Reynolds Averaged Navier- Stokes model. The resulting differences in the overall behaviour of the cell metabolism through the lens of glycolytic oscillations are discussed. In addition, possible pitfalls in model validity such as grid dependence, the effects of heterogeneous particle distributions and the effects of particle numbers were explored. The synchronisation and desynchronisation of glycolytic oscillations as observed in Lapin et al. 2004 were able to be reproduced. ... Read more