(164d) Multifunctional Reactor for Process Intensification of a Multi-Enzymatic Cascade

Biocatalysis plays a crucial role in the sustainable production of bulk chemicals, specialty chemicals, and high-value products. The use of enzymes, can reduce investment and operational costs and decreases the risk of accidents by enabling milder reaction conditions compared to chemical processes [1]. Enzyme-catalyzed reactions also have high selectivity, increasing yields, and reducing the complexity and costs of downstream processing. However, several challenges still exist, such as high enzyme costs, reactant or product inhibition, and low conversion rates. In order to overcome these, multi-enzymatic cascades can be a sufficient option. They allow the production of tailored products with flexible synthesis routes and can be operated without the separation of the intermediate [2]. In addition to the use of enzymes, process intensification is a key factor in improving biocatalytic processes for sustainable production. As stated by Stankiewicz process intensification “consists in the development of innovative apparatuses and techniques that offer drastic improvements in chemical manufacturing and processing, substantially decreasing equipment [..] and ultimately leading to cheaper, safer, technologics” [3]. In biocatalytic processes, this can be achieved through the use of multifunctional reactors, as e.g. achieved in reactive separations. Multifunctional reactors can be designed to overcome limitations such as enzyme instability or low conversion rates by providing suitable reaction conditions for biocatalysts [4]. Furthermore, a membrane bioreactor can be combined with a packed bed reactor to improve mass transfer and minimize substrate inhibition [4,5].

This contribution investigates the intensification of a biocatalytic process for producing cinnamyl cinnamate, a natural flavor and fragrance. The process employs a multi-enzymatic cascade, as depicted in Figure 1. The first step of the cascade converts cinnamyl aldehyde into cinnamyl alcohol. However, the alcohol dehydrogenase (ADH) used in this reaction requires the costly co-factor NADH. To reduce the use of NADH, a second reaction is introduced for co-factor regeneration using a formate dehydrogenase (FDH). Both reactions take place in the aqueous phase The third reaction in the cascade takes place in the organic phase, where an esterification reaction using an immobilized lipase forms the final product, cinnamyl cinnamate. To ensure optimal reaction conditions, two phases are utilized. The esterification reaction cannot occur in the aqueous phase, as water is produced as a byproduct. The ADH and FDH enzymes are highly sensitive to the organic medium, necessitating the use of an aqueous medium. This reaction system offers an opportunity to produce a natural flavor and fragrance sustainably. However, its technical implementation is challenging as it requires controlling three reactions in two phases. This contribution explores the realization of the process in a miniplant scale and the potential for improving the process using multifunctional reactors.

The shown reaction system was operated in a former project in a miniplant. the previous setup of the process, which comprised a reactor for the two aqueous-phase reactions, an extractive centrifuge for facilitating mass transfer between the phases, and a reactor for the organic-phase reaction [6]. Recent simulation studies identified two primary, independent bottlenecks in this setup: the immobilized enzymes in the aqueous phase and the reflux stream of the organic phase from the fixed bed reactor back into the centrifuge during continuous operation.

Set-up

The integrated process with modifications for both bottlenecks is shown in Figure 2. When immobilized on a silica carrier, ADH and FDH exhibited only 4% residual activity. Consequently, an alternative to immobilization that keeps the enzymes in place had to be found. An enzyme membrane reactor was suitable for this purpose since it allowed the reaction medium, substrates, and products to pass through while retaining the native enzymes. This can significantly enhance enzyme activity and reaction velocity. The second part for improving the process was to reduce the reflux rate from the organic reactor into the centrifuge during continuous operation. According to Baldea [7], high reflux rates indicated the need for further process integration, such as the integration of the reactor and the extractive centrifuge as a reactive extraction centrifuge. In order to store the organic volume that was not inside the centrifuge, a tank for the organic phase was added. The subsequent sections will describe the implementation of the enzyme membrane reactor, the reactive extraction centrifuge, and the simulations in a miniplant.

Investigation of membrane reactor and reactive extractive centrifuge

Prior to their integration into the process, both the enzyme membrane reactor and the reactive extraction centrifuge were investigated individually. To establish the feasibility of the enzyme membrane reactor, a solvent resistant membrane that offers a high permeate flux, and provides a suitable cut-off between the enzyme and the product size was required. A screening of seven membranes identified an ultrafiltration Pebax® membrane that met these requirements. This membrane was then integrated into an enzyme membrane reactor, which was used to investigate ADH-catalyzed synthesis. Figure 3 illustrates the experimental results of the enzyme membrane reactor, which reveals a sharp increase in alcohol concentration after a dead time of around four hours. The dead time can be explained by the high volume of the membrane plant compared to the volume flow in and out of the plant. The effects of the enzyme membrane reactor were being compared to those of the previous setup, in which immobilized enzymes were used. The experimental results show that the enzyme membrane reactor produced an intermediate alcohol yield that is about 4 times higher than that achieved using the previous setup in the same time span. Adjusting the startup and operation procedures to reduce the system's dead time is expected to increase this effect further.

To study the reactive extraction centrifuge, a modified extractive centrifuge was used as there are no reactive extraction centrifuges commercially available. The reactive zone was integrated into the rotor of the centrifuge, and operation conditions that allow for clear phase separation were determined without conducting a reaction. The volume flow into the reactive extraction centrifuge and the rotational speed were found to be the key factors that influence phase separation, with a rotational speed of 500 rpm and a total volume flow of 500 mL/min being suitable parameters [8]. The whole reactive extraction centrifuge was then studied, with exemplary results presented in Figure 4. These results demonstrate a clear increase in the final product concentration during the operation time, indicating that the novel, internally constructed apparatus is operational.

Simulation of the integrated process

To gain a deeper understanding of the process, it is beneficial to create a mathematical model. In this study, a dynamic model will be implemented in Python with time discretized in one-minute steps. The developed model includes both phases, as well as the concentrations of all substrates, intermediates, and products. To create the model, the reaction kinetics of all three reactions were determined on a laboratory scale, and physical properties and substance-related data, such as the partition coefficient, were used to describe the process [6]. Solubilities were taken into consideration as well. The concentrations, volumes, and temperatures are limited to be zero or higher as a boundary condition. This model can be used to conduct case studies, particularly to determine the initial substrate concentrations and operational conditions, such as the temperature.

Conclusion

To summarize, this contribution explores how the use of multi-enzymatic cascades and process intensification can help reduce the effect of challenges such as high enzyme costs and low conversion rates. Specifically, the intensification of a biocatalytic process for producing cinnamyl cinnamate and the potential for improving the process using multifunctional reactors is investigated. The study proposes the use of enzyme membrane reactors and reactive extraction centrifuges in the process to increase the efficiency of the reaction and to successfully operate the novel apparatus. In addition to that the process is successfully modeled and simulated in order to gain a better understanding of the complex process. This model can be used for further studies to determine bottlenecks and possibilities for further improvement of the process.

References

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