Process Integration, Assessment, and Online Monitoring of Biotechnological Itaconic Acid Production

  • Prozessintegration, -evaluierung und Online-Monitoring der biotechnologischen Itaconsäureproduktion

Kreyenschulte, Dirk Herbert; Büchs, Jochen (Thesis advisor); Krull, Rainer (Thesis advisor)

Aachen (2016)
Dissertation / PhD Thesis

Dissertation, Rheinisch-Westfälische Technische Hochschule Aachen, 2016


With the gradual establishment of a biobased economy and the imminent switch from petrochemicals to products from renewable resources, the fermentative utilization of sugars and other feedstocks has gained importance within the last years. As one of the most promising value-added chemicals from biomass, itaconic acid has a vast potential in the production of versatile, biobased polymers and the replacement of petrochemical-based materials such as acrylic acid. With regard to biotechnological itaconic acid production, however, new, innovative processes are required to enhance profitability and competitiveness in future markets. Therefore, process integration, assessment, and online monitoring of itaconic acid production were addressed within this thesis. Prior to submerged itaconic acid production, the generation of suitable seed cultures for the cultivation of Aspergillus terreus was investigated. For the most commonly used Czapek-Dox agar medium, a modified formulation was proposed, which reliably induced sporulation without any variability in the observed phenotypes. Subsequently, the cultivation of A. terreus was established in unbaffled shake flasks as well as a laboratory scale bioreactor. Online measurement of the respiratory quotient was found to directly reflect itaconic acid formation. Hence, phases of strong itaconic acid production as well as conditions negatively affecting product formation such as product inhibition could be identified online.To alleviate the negative effect of product inhibition via process integration, the applicability of reactive extraction for in situ product removal was investigated. Initially, the biocompatibility of several solvents was assessed based on their influence on the respiratory activity of A. terreus. As a result, a mixture of the extractant trioctylamine and the diluent isopropyl myristate was chosen for further experiments. As expected, an increase in the amount of trioctylamine increased itaconic acid extraction efficiency. However, the simultaneous increase in pH due to the alkaline properties of trioctylamine attenuated this effect. In addition to reactive extraction, the feasibility of itaconic acid back-extraction into an aqueous trimethylamine solution was confirmed. Finally, reactive extraction was successfully integrated into the cultivation of A. terreus via solvent addition after 63 h. As a consequence of the elevated pH and the reduced amount of itaconic acid, glucose consumption and product formation were considerably enhanced. In combination with an increased amount of glucose either supplied initially or during the cultivation, in situ reactive extraction led to an increase in total itaconic acid concentration from 70 to 105 g/L. Simultaneously, volumetric productivity increased from 0.72 to 0.91 g/L/h. With reactive extraction, therefore, a highly promising method for in situ removal of itaconic acid could be established, which still has great potential for further optimization.During the assessment of the potential profitability and feasibility of new production routes, aerobic fermentation can turn out to be a major factor contributing to the operating expenditure of the overall process. The accurate evaluation of its energy demand is, therefore, of prime importance. To this end, a tool was developed based on established and generally accepted correlations considering relevant parameters and constraints for bioreactor operation. While the assessment is performed for large-scale reactors (10 - 100 m³), it is based on data from small-scale experiments using the oxygen transfer rate as a scale-up criterion. Among the evaluated constraints, the flooding-loading transition of the bioreactor proved to be a crucial criterion for culture volumes of more than 20 m³. Minimum energy demand for cooling, agitation, and aeration could thus be achieved at low agitation and high aeration intensity. At moderate oxygen transfer requirements, bioreactor pressurization was not found to increase energy efficiency of aerobic processes. It was, however, shown to be indispensable for processes with high oxygen demands and elevated foam formation. The tool was applied to assess itaconic acid as well as lysine fermentation processes. Due to considerable differences in oxygen demand, average power consumption was found to be at 0.51 kW/m³ (itaconic acid) and 2.61 kW/m³ (lysine), thus arguing against the utilization of general rule of thumb values. The established tool, therefore, provides an efficient means to specify estimates of the energy demand in consideration of the respective process.For the development of biotechnological processes in academia as well as in industry new techniques are required which enable online monitoring for process characterization and control. Nuclear magnetic resonance (NMR) spectroscopy is a promising analytical tool, which has already found broad applications in offline process analysis. The use of online monitoring, however, is oftentimes constrained by high complexity of custom-made NMR bioreactors and considerable costs for high-field NMR instruments (> US$200,000). Therefore, low-field 1H NMR was investigated in a bypass system for real-time observation of fermentation processes. The new technique was validated with two microbial systems. For the yeast Hansenula polymorpha glycerol consumption could accurately be assessed in spite of the presence of high amounts of complex constituents in the medium. During cultivation of the fungal strain Ustilago maydis, which is accompanied by the formation of several by-products, the concentrations of glucose, itaconic acid and the relative amount of glycolipids could be quantified. While low-field spectra were characterized by reduced spectral resolution compared to high-field NMR, the compact design combined with the high temporal resolution (15 s to 8 min) of spectra acquisition allowed online monitoring of the respective processes. Both applications clearly demonstrate that the investigated technique is well suited for reaction monitoring in opaque media, while at the same time it is highly robust and chemically specific. It can thus be concluded that low-field NMR spectroscopy has a great potential for non-invasive online monitoring of biotechnological processes at the research and practical industrial scales.In conclusion, new concepts and methods were presented for process integration, assessment, and monitoring of biotechnological itaconic acid production. While these aspects as well as the associated methodologies are quite different, it is believed that their combination and consolidation will set the ground for the next generation of itaconic acid production processes.