Ulrich Krühne

Challenges in industrial fermentation technology research

Industrial fermentation processes are increasingly popular, and are considered an important technological asset for reducing our dependence on chemicals and products produced from fossil fuels. However, despite their increasing popularity, fermentation processes have not yet reached the same maturity as traditional chemical processes, particularly when it comes to using engineering tools such as mathematical models and optimization techniques. This perspective starts with a brief overview of these engineering tools. However, the main focus is on a description of some of the most important engineering challenges: scaling up and scaling down fermentation processes, the influence of morphology on broth rheology and mass transfer, and establishing novel sensors to measure and control insightful process parameters. The greatest emphasis is on the challenges posed by filamentous fungi, because of their wide applications as cell factories and therefore their relevance in a White Biotechnology context. Computational fluid dynamics (CFD) is introduced as a promising tool that can be used to support the scaling up and scaling down of bioreactors, and for studying mixing and the potential occurrence of gradients in a tank.

Monitoring and control of microbioreactors: An expert opinion on development needs

This perspective article is based on an expert panel review on microbioreactor applications in biochemical and biomedical engineering that was organized by the M³C (measurement, monitoring, modelling and control) Working Group of the European Section of Biochemical Engineering Science (ESBES) in the European Federation of Biotechnology (EFB). The aim of the panel was to provide an updated view on the present status of the subject and to identify critical needs and issues for furthering the successful development of microbioreactor monitoring and control. This will benefit future bioprocess development and in vitro toxicity testing. The article concludes with a set of recommendations for extended use and further development of microbioreactors.

Microfluidic device for continuous single cells analysis via Raman spectroscopy enhanced by integrated plasmonic nanodimers

In this work a Raman flow cytometer is presented. It consists of a microfluidic device that takes advantages of the basic principles of Raman spectroscopy and flow cytometry. The microfluidic device integrates calibrated microfluidic channels- where the cells can flow one-by-one -, allowing single cell Raman analysis. The microfluidic channel integrates plasmonic nanodimers in a fluidic trapping region. In this way it is possible to perform Enhanced Raman Spectroscopy on single cell. These allow a label-free analysis, providing information about the biochemical content of membrane and cytoplasm of the each cell. Experiments are performed on red blood cells (RBCs), peripheral blood lymphocytes (PBLs) and myelogenous leukemia tumor cells (K562).

Experimental and model assisted investigation of an operational strategy for the BPR under low influent concentrations

The behaviour of a pilot scale biological phosphorus removal process (BPR) of the alternating type was investigated during periods of low influent concentrations and increased hydraulic load. A process disturbance of this type result in an increase in the phosphate concentration level in the anoxic/aerobic reactors and in the plant effluent shortly after the influent wastewater returns to normal strength. The accumulation of phosphorus in the system was avoided by the addition of an external carbon source either to the influent or to the effluent from the anaerobic reactor in form of sodium acetate. With the help of such an addition, the internal carbon storage compounds could be maintained at a high level, which is shown by poly-hydroxy-alcanoates (PHA) measurements. Several levels of acetate addition were investigated experimentally in order to determine a minimal amount of internally stored carbon, which could ensure the stabilization of BPR during such dynamic influent conditions. Furthermore reduction of aeration time during periods of low influent concentrations was investigated. It was observed that BPR was stabilized by combining a reduction of aeration time with carbon source addition, which maintained the internal stored carbon at a higher level. This combined control action resulted in a desired high BPR activity when the normal strength of the influent wastewater was re-established. The failure of the BPR process was sometimes observed even when comparatively high concentrations of PHA could be detected and an identification of a minimal PHA level was not possible. During this investigation an extended version of the activated sludge model No. 2 (ASM2), which includes denitrification by phosphate accumulating organisms, is used for the detailed analysis of the experiments. The model predicted the phosphorus build-up after the process disturbance as well as the performance during the stabilized experiments. Assisted by the model, the investigations indicate that a PHA limitation is not the only factor affecting the recovery of the BPR process during periods of low influent concentrations.

Biocatalytic process development using microfluidic miniaturized systems

Abstract The increasing interest in biocatalytic processes means there is a clear need for a new systematic development paradigm which encompasses both protein engineering and process engineering. This paper argues that through the use of a new microfluidic platform, data can be collected more rapidly and integrated with process modeling, can provide the basis for validating a reduced number of potential processes. The miniaturized platform should use a smaller reagent inventory and make better use of precious biocatalysts. The EC funded BIOINTENSE project will use ω-transaminase based synthesis of chiral amines as a test-bed for assessing the viability of such a high throughput biocatalytic process development, and in this paper, such a vision for the future is presented.

A microfluidic toolbox for the development of in-situ product removal strategies in biocatalysis

A microfluidic toolbox for accelerated development of biocatalytic processes has great potential. This is especially the case for the development of advanced biocatalytic process concepts, where reactors and product separation methods are closely linked together to intensify the process performance, e.g., by the use of in-situ product removal (ISPR). This review provides a general overview of currently available tools in a microfluidic toolbox and how this toolbox can be applied to the development of advanced biocatalytic process concepts. Emphasis is placed on describing the possibilities and advantages of the microfluidic toolbox that are difficult to achieve with conventional batch-processbased technologies. Application of this microfluidic toolbox will potentially make it possible to intensify biocatalytic reactions and thereby facilitate the development towards novel and advanced biocatalytic processes, which in many cases have proven too difficult in conventional batch equipment.

Refractive microlenses produced by excimer laser irradiation of poly (methyl methacrylate)

A method has been developed whereby refractive microlenses can be produced in poly (methyl methacrylate) by excimer laser irradiation at λ = 248 nm. The lenses are formed by a combined photochemical and thermal process. The lenses are formed as depressions in the substrate material (negative focal length), which makes replication necessary in order to obtain lenses with a positive focal length. The method allows for considerable flexibility with respect to the pattern of lenses and the properties of the individual lenses. In this investigation it was possible to vary the diameter of the lenses between 30 and 500 µm and the focal lengths between 300 µm and several mm.

Development of in situ product removal strategies in biocatalysis applying scaled-down unit operations

An experimental platform based on scaled‐down unit operations combined in a plug‐and‐play manner enables easy and highly flexible testing of advanced biocatalytic process options such as in situ product removal (ISPR) process strategies. In such a platform, it is possible to compartmentalize different process steps while operating it as a combined system, giving the possibility to test and characterize the performance of novel process concepts and biocatalysts with minimal influence of inhibitory products. Here the capabilities of performing process development by applying scaled‐down unit operations are highlighted through a case study investigating the asymmetric synthesis of 1‐methyl‐3‐phenylpropylamine (MPPA) using ω‐transaminase, an enzyme in the sub‐family of amino transferases (ATAs). An on‐line HPLC system was applied to avoid manual sample handling and to semi‐automatically characterize ω‐transaminases in a scaled‐down packed‐bed reactor (PBR) module, showing MPPA as a strong inhibitor. To overcome the inhibition, a two‐step liquid–liquid extraction (LLE) ISPR concept was tested using scaled‐down unit operations combined in a plug‐and‐play manner. Through the tested ISPR concept, it was possible to continuously feed the main substrate benzylacetone (BA) and extract the main product MPPA throughout the reaction, thereby overcoming the challenges of low substrate solubility and product inhibition. The tested ISPR concept achieved a product concentration of 26.5 gMPPA · L−1, a purity up to 70% gMPPA · gtot−1 and a recovery in the range of 80% mol · mol−1 of MPPA in 20 h, with the possibility to increase the concentration, purity, and recovery further. Biotechnol. Bioeng. 2017;114: 600–609.

Measurement of oxygen transfer from air into organic solvents

Abstract BACKGROUND The use of non‐aqueous organic media is becoming increasingly important in many biotechnological applications in order to achieve process intensification. Such media can be used, for example, to directly extract poorly water‐soluble toxic products from fermentations. Likewise many biological reactions require the supply of oxygen, most normally from air. However, reliable online measurements of oxygen concentration in organic solvents (and hence oxygen transfer rates from air to the solvent) has to date proven impossible due to limitations in the current analytical methods. RESULTS For the first time, online oxygen measurements in non‐aqueous media using a novel optical sensor are demonstrated. The sensor was used to measure oxygen concentration in various organic solvents including toluene, THF, isooctane, DMF, heptane and hexane (which have all been shown suitable for several biological applications). Subsequently, the oxygen transfer rates from air into these organic solvents were measured. CONCLUSION The measurement of oxygen transfer rates from air into organic solvents using the dynamic method was established using the solvent resistant optical sensor. The feasibility of online oxygen measurements in organic solvents has also been demonstrated, paving the way for new opportunities in process control.

Multi-function microfluidic platform for sensor integration

The limited availability of metabolite-specific sensors for continuous sampling and monitoring is one of the main bottlenecks contributing to failures in bioprocess development. Furthermore, only a limited number of approaches exist to connect currently available measurement systems with high throughput reactor units. This is especially relevant in the biocatalyst screening and characterization stage of process development. In this work, a strategy for sensor integration in microfluidic platforms is demonstrated, to address the need for rapid, cost-effective and high-throughput screening in bioprocesses. This platform is compatible with different sensor formats by enabling their replacement and was built in order to be highly flexible and thus suitable for a wide range of applications. Moreover, this re-usable platform can easily be connected to analytical equipment, such as HPLC, laboratory scale reactors or other microfluidic chips through the use of standardized fittings. In addition, the developed platform includes a two-sensor system interspersed with a mixing channel, which allows the detection of samples that might be outside the first sensors range of detection, through dilution of the sample solution up to 10 times. In order to highlight the features of the proposed platform, inline monitoring of glucose levels is presented and discussed. Glucose was chosen due to its importance in biotechnology as a relevant substrate. The platform demonstrated continuous measurement of substrate solutions for up to 12 h. Furthermore, the influence of the fluid velocity on substrate diffusion was observed, indicating the need for in-flow calibration to achieve a good quantitative output.