Anita Šalić

Modeling and kinetic parameter estimation of alcohol dehydrogenase-catalyzed hexanol oxidation in a microreactor

A mathematical model for hexanol oxidation catalyzed by NAD+‐dependent alcohol dehydrogenase from bakers yeast in a microreactor was developed and compared with the model when the reaction takes place in a macroscopic reactor. The enzyme kinetics was modeled as a pseudo‐homogeneous process with the double substrate Michaelis–Menten rate expression. In comparison with the kinetic parameters estimated in the cuvette, a 30‐fold higher maximum reaction rate and a relatively small change in the saturation constants are observed for the kinetic parameters estimated in the continuously operated tubular microreactor (Vm1=197.275 U/mg, Kmhexanol=9.420 mmol/L, and Km1NAD+=0.187 mmol/L). Kinetic measurements performed in the microreactor, estimated from the initial reaction rate experiments at the residence time of 36 s, showed no product inhibition, which could be explained by hydrodynamic effects and the continuous removal of inhibiting products. The Fourier amplitude sensitivity test method was applied for global kinetic parameter analysis, which shows a significant increase in the sensitivity of Km1NAD+ in the microreactor. Independent experiments performed in the microreactor were used to validate and to verify the developed mathematical model.

Bioproduction of Food Additives Hexanal and Hexanoic Acid in a Microreactor

Hexanal and hexanoic acid have number of applications in food and cosmetic industry because of their organoleptic characteristics. Problems like low yields, formation of unwanted by-products, and large quantities of waste in their traditional production processes are the reasons for developing new production methods. Biotransformation in a microreactor, as an alternative to classical synthesis processes, is being investigated. Because conditions in microreactors can be precisely controlled, the quality of the product and its purity can also be improved. Biocatalytic oxidation of hexanol to hexanal and hexanoic acid using suspended and immobilized permeabilized whole baker’s yeast cells and suspended and immobilized purified alcohol dehydrogenase (ADH) was investigated in this study. Three different methods for covalent immobilization of biocatalyst were analyzed, and the best method for biocatalyst attachment on microchannel wall was used in the production of hexanal and hexanoic acid.

Enzyme-catalysed Biodiesel Production from Edible and Waste Cooking Oils

Biodiesel synthesis was performed as transesterification of edible and waste cooking sunflower oil catalysed by free lipase from Thermomyces lanuginosus (Lipolase 100L). Experiments were performed at three different temperatures (T = 40, 50 and 60 °C) as one-step and four-step reactions with methanol. The highest fatty acids methyl esters (FAME) content (C = 95 %) was achieved in the one-step transesterification reaction of edible sunflower oil performed at 40 °C.

Catechol removal from aqueous media using laccase immobilized in different macro- and microreactor systems

Laccase belongs to the group of enzymes that are capable to catalyze the oxidation of phenols. Since the water is only by-product in laccase-catalyzed phenol oxidations, it is ideally “green” enzyme with many possible applications in different industrial processes. To make the oxidation process more sustainable in terms of biocatalyst consumption, immobilization of the enzyme is implemented in to the processes. Additionally, when developing a process, choice of a reactor type plays a significant role in the total outcome.In this study, the use of immobilized laccase from Trametes versicolor for biocatalytic catechol oxidation was explored. Two different methods of immobilization were performed and compared using five different reactor types. In order to compare different systems used for catechol oxidation, biocatalyst turnover number and turnover frequency were calculated. With low consumption of the enzyme and good efficiency, obtained results go in favor of microreactors with enzyme covalently immobilized on the microchannel surface.

NADH oxidation in a microreactor with an oscillating magnetic field

In this study, magnetic nanoparticles (MNPs) of maghemite (γ-Fe2O3) were synthesized and characterized. The method of multifactor experimental design and evolutionary operation (EVOP) was used to optimize immobilization of the alcohol dehydrogenase (ADH) enzyme on MNPs. Optimal operating conditions for the immobilization process were determined (γADH = 0.08 mg/mL, 2% glutaraldehyde for surface activation, t = 28 h), and in such conditions, a specific activity of S.A. = 118 ± 6 U/mg and immobilization efficiency of η = 84.97 ± 3.67% were achieved. Compared to the native enzyme, ADH immobilized on MNPs retained 66.45 ± 3.66% of the initial activity. ADH immobilized on MNPs at optimal conditions was used as a biocatalyst for model reaction—NADH oxidation. NADH oxidation was performed in two different magnetic microreactor configurations: (1) microreactor equipped with permanent square magnets and (2) microreactor equipped with an electromagnet and an oscillating magnetic field that enables magnetic particles movement in the microreactor. In the system with the oscillating magnetic field, equal conversion (X = 100%) was achieved in 2-fold shorter residence time.

ADH-catalysed hexanol oxidation with fully integrated NADH regeneration performed in microreactors connected in series

Hexanal is produced by the oxidation of hexanol using NADH dependent alcohol dehydrogenase (ADH). Coenzyme, NADH regeneration is needed in order to make the ADH-catalysed process more sustainable. In this investigation, the coenzyme regeneration was catalysed by the same enzyme, ADH, which catalysed the main reaction, i.e., the oxidation of hexanol. Different sources of ADH were studied (suspended and immobilized enzyme ADH and permeabilized bakers yeast cells) to find the optimal catalyst. The best results were obtained by using suspended enzyme, where 100% conversion of the coenzyme was achieved with a very short residence time (τ = 0.8 s). The final phase of investigation was development of an integrated system with two microreactor chips connected in series. The first chip was used for hexanol oxidation and the second for the simultaneous coenzyme regeneration. Regenerated coenzyme was reused by recirculation in to the first chip, where the oxidation step was continuously performed for three days without the need for the addition of fresh coenzyme.

NADH oxidation in a microreactor catalysed by ADH immobilised on γ-Fe2O3 nanoparticles

Abstract A new concept of nicotinamide adenine dinucleotide hydrate (NADH) oxidation which combines advantages of the microreactor technology with the advantages of magnetic nanoparticles (MNPs) application is developed. Acetaldehyde was used as a substrate for the NADH regeneration process while the reaction was performed in a batch reactor and in a microreactor using alcohol dehydrogenase (ADH)-loaded MNPs. Three different microreactor systems with MNPs were studied, two with stationary MNPs trapped on the inner surface of microchannel by permanent magnetic field and one where the MNPs actively moved across the channel (movement inside microchannel allowed by an oscillating magnetic field). In a reactor system with an oscillating magnetic field and an actively moving ADH-loaded MNPs 100% NADH conversion was achieved for residence time of just 2 min.

Lipase catalysed biodiesel synthesis with integrated glycerol separation in continuously operated microchips connected in series

Although the application of microreactors in different processes has been extensively explored in recent decades, microreactors continue to be underexplored in the context of the enzyme-catalysed process for biodiesel production. Due to their numerous advantages, microreactors could become the next step in the development of a biodiesel production process characterised by sustainability, cost-effectiveness and energy efficiency. In this investigation, biodiesel production was catalysed by lipase from Thermomyces lanuginosus (Lipolase L100). Edible sunflower oil was used as a model substrate in order to investigate the process. After optimal process conditions had been determined, waste-cooking oil was used for biodiesel production to make the production process more sustainable. Three different substrate-feeding strategies were investigated and finally an optimal strategy was proposed. In all the investigated systems, fatty acids methyl esters (FAME) content was higher than 95% and obtained in a significantly shorter time (less than 2 h) compared to the batch process in which biodiesel production was catalysed by lipase (C = 95%, t = 96 h). After the optimal biodiesel production system had been proposed, an integrated system with two microchips connected in series was developed. The first microchip was used for biodiesel production and the second for simultaneous purification i.e. glycerol separation. Finally, purified biodiesel was produced with glycerol content below the detection limit.

Mathematical modelling of polyphenol extraction by aqueous two-phase system in continuously operated macro- and micro-extractors

ABSTRACT An aqueous two-phase system (ATPS) in combination with macro- and micro-extractors was used for polyphenol extraction from a model solution (gallic acid) and three real samples (red and white wine, and orange juice). The objective of the present study was to apply a mathematical model that successfully describes and predicts performances of macro- and micro-extractors. The micro-extractor system was selected as the most suitable for the polyphenol extraction because the same extraction efficiency was obtained for two levels of magnitude shorter residence time compared to the macro-extractor. Based on good agreement between model predictions and experimental results, the obtained simulations could be used for further process optimization and improvement.

Introduction to environmental engineering

Abstract Nowadays we can easily say that environmental engineering is truly an interdisciplinary science. Combining biology, ecology, geology, geography, mathematics, chemistry, agronomy, medicine, economy, etc. environmental engineering strives to use environmental understanding and advancements in technology to serve mankind by decreasing production of environmental hazards and the effects of those hazards already present in the soil, water, and air. Major activities of environmental engineer involve water supply, waste water and solid management, air and noise pollution control, environmental sustainability, environmental impact assessment, climate changes, etc. And all this with only one main goal – to prevent or reduce undesirable impacts of human activities on the environment. To ensure we all have tomorrow.