Activity-Based Models to Predict Kinetics of Levulinic Acid Esterification.

The solvent is of prime importance in biomass conversion as it influences dissolution, reaction kinetics, catalyst activity and thermodynamic equilibrium of the reaction system. So far, activity-based models were developed to predict kinetics and equilibria, but the influence of the catalyst on kinetics has not been succesfully predicted by thermodynamic models. In this work, the thermodynamic model ePC-SAFT advanced was used to predict the activities of the reactants and of the catalyst at various conditions (temperature, reactant concentrations, γ-valerolactone GVL cosolvent addition, catalyst concentration) for the homogeneously acid-catalyzed esterification of levulinic acid (LA) with ethanol. Different kinetic models were applied, and it was found that the catalyst influence on kinetics could be predicted correctly by simultaneously solving the dissociation equilibrium of H2 SO4 catalyst along the reaction coordinate and by relating reaction kinetics to proton activity. ePC-SAFT advanced model parameters were only fitted to reaction-independent phase equilibrium data. The key reaction properties were determined by applying ePC-SAFT advanced to one experimental kinetic curve for a set of temperatures, yielding the reaction enthalpy at standard state Δ R H 0 = 11 . 48 k J m o l - 1 ${{\Delta }^{R}{H}^{0}=11.48\ kJ\ mo{l}^{-1}}$ , activation energy E A = 30 . 28 k J m o l - 1 ${{E}_{A}=30.28\ kJ\ mo{l}^{-1}}$ and the intrinsic reaction rate constant k=0.011 s-1 at 323 K, which is independent of catalyst concentration. The new procedure allowed an a-priori identification of the effects of catalyst, solvent and reactant concentration on LA esterification.

Simultaneous Predictions of Chemical and Phase Equilibria in Systems with an Esterification Reaction Using PC-SAFT.

The study of chemical reactions in multiple liquid phase systems is becoming more and more relevant in industry and academia. The ability to predict combined chemical and phase equilibria is interesting from a scientific point of view but is also crucial to design innovative separation processes. In this work, an algorithm to perform the combined chemical and liquid-liquid phase equilibrium calculation was implemented in the PC-SAFT framework in order to predict the thermodynamic equilibrium behavior of two multicomponent esterification systems. Esterification reactions involve hydrophobic reacting agents and water, which might cause liquid-liquid phase separation along the reaction coordinate, especially if long-chain alcoholic reactants are used. As test systems, the two quaternary esterification systems starting from the reactants acetic acid + 1-pentanol and from the reactants acetic acid + 1-hexanol were chosen. It is known that both quaternary systems exhibit composition regions of overlapped chemical and liquid-liquid equilibrium. To the best of our knowledge, this is the first time that PC-SAFT was used to calculate simultaneous chemical and liquid-liquid equilibria. All the binary subsystems were studied prior to evaluating the predictive capability of PC-SAFT toward the simultaneous chemical equilibria and phase equilibria. Overall, PC-SAFT proved its excellent capabilities toward predicting chemical equilibrium composition in the homogeneous composition range of the investigated systems as well as liquid-liquid phase behavior. This study highlights the potential of a physical sound model to perform thermodynamic-based modeling of chemical reacting systems undergoing liquid-liquid phase separation.

Carboxylation of Acetylene without Salt Waste: Green Synthesis of C4 Chemicals Enabled by a CO2 -Pressure Induced Acidity Switch.

The inherent formation of salt waste in C-H carboxylations is a key obstacle precluding the utilization of CO2 as C1 building block in the industrial synthesis of base chemicals. This challenge is addressed in a circular process for the production of the C4 base chemical dimethyl succinate from CO2 and acetylene. At moderate CO2 pressures, acetylene is doubly carboxylated in the presence of cesium carbonate. Hydrogenation of the C-C triple bond stabilizes the product against decarboxylation. By increasing the CO2 pressure to 70 bar, the medium is reversibly acidified, allowing an esterification of the succinate salt with methanol. The cesium base and the hydrogenation catalyst are regenerated and can be reused. This provides the proof of concept for a salt-free route to C4 chemicals from biogas (CH4 /CO2 ). The origin of this reversible acidity switch and the critical roles of the cesium base and the NMP/MeOH solvents were elucidated by thermodynamic modeling.

Osmolyte effect on enzymatic stability and reaction equilibrium of formate dehydrogenase.

Osmolytes are well-known biocatalyst stabilisers as they promote the folded state of proteins, and a stabilised biocatalyst might also improve reaction kinetics. In this work, the influence of four osmolytes (betaine, glycerol, trehalose, and trimethylamine N-oxide) on the activity and stability of Candida bondinii formate dehydrogenase cbFDH was studied experimentally and theoretically. Scanning differential fluorimetric studies were performed to assess the thermal stability of cbFDH, while UV detection was used to reveal changes in cbFDH activity and reaction equilibrium at osmolyte concentrations between 0.25 and 1 mol kg-1. The thermodynamic model ePC-SAFT advanced allowed predicting the effects of osmolyte on the reaction equilibrium by accounting for interactions involving osmolyte, products, substrates, and water. The results show that osmolytes at low concentrations were beneficial for both, thermal stability and cbFDH activity, while keeping the equilibrium yield at high level. Molecular dynamics simulations were used to describe the solvation around the cbFDH surface and the volume exclusion effect, proofing the beneficial effect of the osmolytes on cbFDH activity, especially at low concentrations of trimethylamine N-oxide and betaine. Different mechanisms of stabilisation (dependent on the osmolyte) show the importance of studying solvent-protein dynamics towards the design of optimised biocatalytic processes.

Make or break: the thermodynamic equilibrium of polyphosphate kinase-catalysed reactions.

Polyphosphate kinases (PPKs) have become popular biocatalysts for nucleotide 5'-triphosphate (NTP) synthesis and regeneration. Two unrelated families are described: PPK1 and PPK2. They are structurally unrelated and use different catalytic mechanisms. PPK1 enzymes prefer the usage of adenosine 5'-triphosphate (ATP) for polyphosphate (polyP) synthesis while PPK2 enzymes favour the reverse reaction. With the emerging use of PPK enzymes in biosynthesis, a deeper understanding of the enzymes and their thermodynamic reaction course is of need, especially in comparison to other kinases. Here, we tested four PPKs from different organisms under the same conditions without any coupling reactions. In comparison to other kinases using phosphate donors with comparably higher phosphate transfer potentials that are characterised by reaction yields close to full conversion, the PPK-catalysed reaction reaches an equilibrium in which about 30% ADP is left. These results were obtained for PPK1 and PPK2 enzymes, and are supported by theoretical data on the basic reaction. At high concentrations of substrate, the different kinetic preferences of PPK1 and PPK2 can be observed. The implications of these results for the application of PPKs in chemical synthesis and as enzymes for ATP regeneration systems are discussed.

Perspective: Increasing blue carbon around Antarctica is an ecosystem service of considerable societal and economic value worth protecting.

Precautionary conservation and cooperative global governance are needed to protect Antarctic blue carbon: the world's largest increasing natural form of carbon storage with high sequestration potential. As patterns of ice loss around Antarctica become more uniform, there is an underlying increase in carbon capture-to-storage-to-sequestration on the seafloor. The amount of carbon captured per unit area is increasing and the area available to blue carbon is also increasing. Carbon sequestration could further increase under moderate (+1°C) ocean warming, contrary to decreasing global blue carbon stocks elsewhere. For example, in warmer waters, mangroves and seagrasses are in decline and benthic organisms are close to their physiological limits, so a 1°C increase in water temperature could push them above their thermal tolerance (e.g. bleaching of coral reefs). In contrast, on the basis of past change and current research, we expect that Antarctic blue carbon could increase by orders of magnitude. The Antarctic seafloor is biophysically unique and the site of carbon sequestration, the benthos, faces less anthropogenic disturbance than any other ocean continental shelf environment. This isolation imparts both vulnerability to change, and an avenue to conserve one of the world's last biodiversity refuges. In economic terms, the value of Antarctic blue carbon is estimated at between £0.65 and £1.76 billion (~2.27 billion USD) for sequestered carbon in the benthos around the continental shelf. To balance biodiversity protection against society's economic objectives, this paper builds on a proposal incentivising protection by building a 'non-market framework' via the 2015 Paris Agreement to the United Nations Framework Convention on Climate Change. This could be connected and coordinated through the Antarctic Treaty System to promote and motivate member states to value Antarctic blue carbon and maintain scientific integrity and conservation for the positive societal values ingrained in the Antarctic Treaty System.

Measurement and modelling solubility of amino acids and peptides in aqueous 2-propanol solutions.

Amino acids and peptides are essential components in the biochemical industry. The final products are employed in a wide range of applications and are often synthesized by fermentation and purified in a complex downstream process. One possible separation step is using an additional solvent to lower the solubility of the desired product and, thus, promote the crystallization of the particular component. Therefore, it is crucial to have accurate knowledge of the solubility of these components. In this work, the solubilities of 20 proteinogenic amino acids and 21 peptides in aqueous 2-propanol solutions were gravimetrically determined. Additionally, the pH values of the saturated liquid phases were measured and the crystal structures of solid crystals were analysed using X-ray diffraction. The anti-solvent 2-propanol caused a decrease in the solubilities of the amino acids and peptides upon increasing its mass fraction. Exceptions were found for amino acids with aromatic substituents, l-phenylalanine and l-tyrosine. The solubility of 15 amino acids and 18 peptides was successfully modelled using the equation of state PC-SAFT that used recently determined melting properties of the amino acids and peptides as input data.

Unravelling the nature of citric acid:L-arginine:water mixtures: the bifunctional role of water.

The use of water as a component of deep eutectic systems (DES) has raised some questions regarding its influence on the nature of the mixture. Does it form a DES or an aqueous solution and what is the role of water? In this work, the nature of citric acid:l-arginine:water mixtures was explored through phase equilibria studies and spectroscopic analysis. In a first step, PC-SAFT was validated as a predictive tool to model the water influence on the solid liquid equilibria (SLE) of the DES reline using the individual-component approach. Hence, activity coefficients in the ternary systems citric acid:l-arginine:water and respective binary combinations were studied and compared using ePC-SAFT. It was observed that the water-free mixtures citric acid:l-arginine showed positive deviation from Raoult's law, while upon addition of water strong negative deviation from Raoult's law was found, yielding melting depressions around 100 K. Besides these strong interactions, pH was found to become acidic (pH = 3.5) upon water addition, which yields the formation of charged species ([H2Cit]- and [l-arg]+). Thus, the increased interactions between the molecules upon water addition might be caused by several mechanisms such as hydrogen bonding or ionic forces, both being induced by water. For further investigation, the liquid mixtures citric acid:l-arginine:water were studied by FTIR and NMR spectroscopy. FTIR spectra disproved a possible solubility enhancement caused by salt formation between citric acid and l-arginine, while NMR spectra supported the formation of a hydrogen bonding network different from the binary systems citric acid:water and l-arginine:water. Either being a DES or other type of non-ideal solution, the liquefaction of the studied systems is certainly caused by a water-mediator effect based on the formation of charged species and cross interactions between the mixture constituents.

Evolution and phylogeny of the deep-sea isopod families Desmosomatidae Sars, 1897 and Nannoniscidae Hansen, 1916 (Isopoda: Asellota).

In the deep sea, the phylogeny and biogeography of only a few taxa have been well studied. Although more than 200 species in 32 genera have been described for the asellote isopod families Desmosomatidae Sars, 1897 and Nannoniscidae Hansen, 1916 from all ocean basins, their phylogenetic relationships are not completely understood. There is little doubt about the close relationship of these families, but the taxonomic position of a number of genera is so far unknown. Based on a combined morphological phylogeny using the Hennigian method with a dataset of 107 described species and a molecular phylogeny based on three markers (COI, 16S, and 18S) with 75 species (most new to science), we could separate Desmosomatidae and Nannoniscidae as separate families. However, we could not support the concept of the subfamilies Eugerdellatinae Hessler, 1970 and Desmosomatinae Hessler, 1970. Most genera of both families were well supported, but several genera appear as para- or even polyphyletic. Within both families, convergent evolution and analogies caused difficulty in defining apomorphies for phylogenetic reconstructions and this is reflected in the results of the concatenated molecular tree. There is no biogeographic pattern in the distribution as the genera occur over the entire Atlantic and Pacific Ocean, showing no specific phylogeographical pattern. Poor resolution at deep desmosomatid nodes may reflect the long evolutionary history of the family and rapid evolutionary radiations. Supplementary Information: The online version contains supplementary material available at 10.1007/s13127-021-00509-9.

Thermodynamics and Kinetics of Glycolytic Reactions. Part I: Kinetic Modeling Based on Irreversible Thermodynamics and Validation by Calorimetry.

In systems biology, material balances, kinetic models, and thermodynamic boundary conditions are increasingly used for metabolic network analysis. It is remarkable that the reversibility of enzyme-catalyzed reactions and the influence of cytosolic conditions are often neglected in kinetic models. In fact, enzyme-catalyzed reactions in numerous metabolic pathways such as in glycolysis are often reversible, i.e., they only proceed until an equilibrium state is reached and not until the substrate is completely consumed. Here, we propose the use of irreversible thermodynamics to describe the kinetic approximation to the equilibrium state in a consistent way with very few adjustable parameters. Using a flux-force approach allowed describing the influence of cytosolic conditions on the kinetics by only one single parameter. The approach was applied to reaction steps 2 and 9 of glycolysis (i.e., the phosphoglucose isomerase reaction from glucose 6-phosphate to fructose 6-phosphate and the enolase-catalyzed reaction from 2-phosphoglycerate to phosphoenolpyruvate and water). The temperature dependence of the kinetic parameter fulfills the Arrhenius relation and the derived activation energies are plausible. All the data obtained in this work were measured efficiently and accurately by means of isothermal titration calorimetry (ITC). The combination of calorimetric monitoring with simple flux-force relations has the potential for adequate consideration of cytosolic conditions in a simple manner.

Thermodynamics and Kinetics of Glycolytic Reactions. Part II: Influence of Cytosolic Conditions on Thermodynamic State Variables and Kinetic Parameters.

For systems biology, it is important to describe the kinetic and thermodynamic properties of enzyme-catalyzed reactions and reaction cascades quantitatively under conditions prevailing in the cytoplasm. While in part I kinetic models based on irreversible thermodynamics were tested, here in part II, the influence of the presumably most important cytosolic factors was investigated using two glycolytic reactions (i.e., the phosphoglucose isomerase reaction (PGI) with a uni-uni-mechanism and the enolase reaction with an uni-bi-mechanism) as examples. Crowding by macromolecules was simulated using polyethylene glycol (PEG) and bovine serum albumin (BSA). The reactions were monitored calorimetrically and the equilibrium concentrations were evaluated using the equation of state ePC-SAFT. The pH and the crowding agents had the greatest influence on the reaction enthalpy change. Two kinetic models based on irreversible thermodynamics (i.e., single parameter flux-force and two-parameter Noor model) were applied to investigate the influence of cytosolic conditions. The flux-force model describes the influence of cytosolic conditions on reaction kinetics best. Concentrations of magnesium ions and crowding agents had the greatest influence, while temperature and pH-value had a medium influence on the kinetic parameters. With this contribution, we show that the interplay of thermodynamic modeling and calorimetric process monitoring allows a fast and reliable quantification of the influence of cytosolic conditions on kinetic and thermodynamic parameters.

Thermophysical properties of glyceline-water mixtures investigated by molecular modelling.

The effect of water content on the static and dynamic properties of the deep eutectic solvent glyceline is studied using molecular dynamics (MD) simulations. Static properties are additionally calculated using the PC-SAFT equation of state. Force fields calibrated on water-free glyceline show predictive power for density and water activity over the entire composition range. In contrast, the PC-SAFT approach using pseudo one-component or two-component modelling strategies performed better for the density or the water activity, respectively. The MD simulations show that at low water content, the hydrogen-bond network between glycerol molecules as well as between glycerol and the cholinium cation is hardly affected by the water molecules while at higher water content, glycerol-glycerol hydrogen bonds are replaced by glycerol-water hydrogen bonds indicating the formation of an aqueous solution accompanied by a strong decrease of the shear viscosity. At the same time, the thermodynamic activity of water increases such that the MD simulations are able to guide the optimal composition with respect to requirements in biocatalytic applications such as low viscosity and low water activity. The combined application of PC-SAFT to efficiently predict static properties and molecular dynamics simulations to predict static and dynamic properties offers a powerful framework in solvent design applications.

Combined co-solvent and pressure effect on kinetics of a peptide hydrolysis: an activity-based approach.

The application of co-solvents and high pressure has been reported to be an efficient means to tune the kinetics of enzyme-catalyzed reactions. Co-solvents and pressure can lead to increased reaction rates without sacrificing enzyme stability, while temperature and pH operation windows are generally very narrow. Quantitative prediction of co-solvent and pressure effects on enzymatic reactions has not been successfully addressed in the literature. Herein, we are introducing a thermodynamic approach that is based on molecular interactions in the form of activity coefficients of substrate and of enzyme in the multi-component solution. This allowed us to quantitatively predict the combined effect of co-solvent and pressure on the kinetic constants, i.e. the Michaelis constant KM and the catalytic constant kcat, of an α-CT-catalyzed peptide hydrolysis reaction. The reaction was studied in the presence of different types of co-solvents and at pressures up to 2 kbar, and quantitative predictions could be obtained for KM, kcat, and finally even primary Michaelis-Menten plots using activity coefficients provided by the thermodynamic model PC-SAFT.

Prediction and Experimental Validation of Co-Solvent Influence on Michaelis Constants: A Thermodynamic Activity-Based Approach.

Co-solvents are known to influence the Michaelis constant K M of enzyme-catalyzed reactions. In the literature, co-solvent effects on K M are usually explained by interactions between enzyme and co-solvent. Very recent works replaced substrate concentrations with thermodynamic activities to separate enzyme-co-solvent from substrate-co-solvent interactions This yields the thermodynamic-activity-based Michalis constant K M a . In this work, this approach was extended to alcohol dehydrogenase (ADH)-catalyzed reduction of acetophenone (ACP), a two-substrate reaction. It was experimentally found that polyethylene glycol (PEG) 6000 increased K M of ACP and decreased K M of nicotinamide adenine dinucleotide (NADH). To predict K M a values, non-covalent interactions between substrates and reaction media were taken into account by electrolyte perturbed-chain statistical associating fluid theory (ePC-SAFT) modelling. In contrast to experimental K M values, their activity-based pendants K M a were independent of co-solvent. To further verify the approach, the reduction of 2-pentanone catalyzed by the same ADH was investigated. Interestingly, the addition of PEG caused a decrease of both K M of 2-pentanone and K M of NADH. Based on K M a values obtained from K M in co-solvent-free conditions and activity coefficients from ePC-SAFT, the influence of the co-solvent on K M was quantitatively predicted. Thus, the approach known for pseudo one-substrate reactions was successfully transferred to two-substrate reactions. Furthermore, the advantage of thermodynamic activities over concentrations in the field of enzyme kinetics is highlighted.

A Distinct Mitochondrial Genome with DUI-Like Inheritance in the Ocean Quahog Arctica islandica.

Mitochondrial DNA (mtDNA) is strictly maternally inherited in metazoans. The major exception to this rule has been found in many bivalve species which allow the presence of different sex-linked mtDNA molecules. This mechanism, named doubly uniparental inheritance (DUI), is characterized by the presence of two mtDNAs: The female mtDNA is found in somatic tissue and female gonads, whereas the male mtDNA is usually found in male gonads and sperm. In this study we highlight the existence of two divergent mitochondrial haplotypes with a low genetic difference around 6-8% in Arctica islandica, a long-lived clam belonging to the Arcticidae, a sister group to the Veneridae in which DUI has been found. Phylogenetic analysis on cytochrome b and 16S sequences from somatic and gonadic tissues of clams belonging to different populations reveals the presence of the "divergent" type in male gonads only and the "normal" type in somatic tissues and female gonads. This peculiar segregation of divergent mtDNA types speaks for the occurrence of the DUI mechanism in A. islandica. This example also highlights the difficulties to assess the presence of such particular mitochondrial inheritance system and underlines the possible misinterpretations in phylogeographic and phylogenetic studies of bivalve species linked to the presence of two poorly differentiated mitochondrial genomes.

Global phylogeography of Oithona similis s.l. (Crustacea, Copepoda, Oithonidae) - A cosmopolitan plankton species or a complex of cryptic lineages?

Traditionally, many small-sized copepod species are considered to be widespread, bipolar or cosmopolitan. However, these large-scale distribution patterns need to be re-examined in view of increasing evidence of cryptic and pseudo-cryptic speciation in pelagic copepods. Here, we present a phylogeographic study of Oithona similis s.l. populations from the Arctic Ocean, the Southern Ocean and its northern boundaries, the North Atlantic and the Mediterrranean Sea. O. similis s.l. is considered as one of the most abundant species in temperate to polar oceans and acts as an important link in the trophic network between the microbial loop and higher trophic levels such as fish larvae. Two gene fragments were analysed: the mitochondrial cytochrome oxidase c subunit I (COI), and the nuclear ribosomal 28 S genetic marker. Seven distinct, geographically delimitated, mitochondrial lineages could be identified, with divergences among the lineages ranging from 8 to 24%, thus representing most likely cryptic or pseudocryptic species within O. similis s.l. Four lineages were identified within or close to the borders of the Southern Ocean, one lineage in the Arctic Ocean and two lineages in the temperate Northern hemisphere. Surprisingly the Arctic lineage was more closely related to lineages from the Southern hemisphere than to the other lineages from the Northern hemisphere, suggesting that geographic proximity is a rather poor predictor of how closely related the clades are on a genetic level.

Crowders and Cosolvents-Major Contributors to the Cellular Milieu and Efficient Means to Counteract Environmental Stresses.

The free energy and conformational landscape of biomolecular systems as well as biochemical reactions depend not only on temperature and pressure, but also on the particular solution conditions. Such conditions include the effects of cosolvents (for example osmolytes) and macromolecular crowding, which are crucial components to understand the energetics and kinetics of biological processes in living system. Such conditions are also important for the understanding of many debilitating diseases, such as those where misfolding and amyloid formation of proteins are involved. Moreover, understanding their effects on biomolecular processes is prerequisite for designing industrially relevant enzymatic reactions, which seldom take place under neat conditions. Here, we review and discuss experimental and theoretical studies on the characterization of cosolvent and crowding induced effects in biologically relevant systems, approaching even the complexity of living organisms. In particular, we focus on cosolvent and crowding effects on the conformational equilibrium and folding kinetics of proteins and nucleic acids as well as on enzymatic reactions, including their effects on the temperature and pressure dependence of these processes. By presenting a few representative examples, we show how such effects are unveiled and described in thermodynamic and kinetic terms.

Different feeding strategies in Antarctic scavenging amphipods and their implications for colonisation success in times of retreating glaciers.

BACKGROUND: Scavenger guilds are composed of a variety of species, co-existing in the same habitat and sharing the same niche in the food web. Niche partitioning among them can manifest in different feeding strategies, e.g. during carcass feeding. In the bentho-pelagic realm of the Southern Ocean, scavenging amphipods (Lysianassoidea) are ubiquitous and occupy a central role in decomposition processes. Here we address the question whether scavenging lysianassoid amphipods employ different feeding strategies during carcass feeding, and whether synergistic feeding activities may influence carcass decomposition. To this end, we compared the relatively large species Waldeckia obesa with the small species Cheirimedon femoratus, Hippomedon kergueleni, and Orchomenella rotundifrons during fish carcass feeding (Notothenia spp.). The experimental approach combined ex situ feeding experiments, behavioural observations, and scanning electron microscopic analyses of mandibles. Furthermore, we aimed to detect ecological drivers for distribution patterns of scavenging amphipods in the Antarctic coastal ecosystems of Potter Cove. In Potter Cove, the climate-driven rapid retreat of the Fourcade Glacier is causing various environmental changes including the provision of new marine habitats to colonise. While in the newly ice-free areas fish are rare, macroalgae have already colonised hard substrates. Assuming that a temporal dietary switch may increase the colonisation success of the most abundant lysianassoids C. femoratus and H. kergueleni, we aimed to determine their consumption rates (g food x g amphipods-1 x day-1) and preferences of macroalgae and fish. RESULTS: We detected two functional groups with different feeding strategies among scavenging amphipods during carcass feeding: carcass 'opener' and 'squeezer'. Synergistic effects between these groups were not statistically verified under the conditions tested. C. femoratus switched its diet when fish was not available by consuming macroalgae (about 0.2 day-1) but preferred fish by feeding up to 80% of its own mass daily. Contrary, H. kergueleni rejected macroalgae entirely and consumed fish with a maximal rate of 0.8 day-1. CONCLUSION: This study reveals functional groups in scavenging shallow-water amphipods and provides new information on coastal intraguild niche partitioning. We conclude that the dietary flexibility of C. femoratus is a potential ecological driver and central to its success in the colonisation of newly available ice-free Antarctic coastal habitats.

Thermodynamics of enzyme-catalyzed esterifications: I. Succinic acid esterification with ethanol.

Succinic acid (SA) was esterified with ethanol using Candida antarctica lipase B immobilized on acrylic resin at 40 and 50 °C. Enzyme activity in the reaction medium was assured prior to reaction experiments. Reaction-equilibrium experiments were performed for varying initial molalities of SA and water in the reaction mixtures. This allowed calculating the molality-based apparent equilibrium constant K m as function of concentration and temperature. K m was shown to depend strongly on the molality of water and SA as well as on temperature. It could be concluded that increasing the molality of SA shifted the reaction equilibrium towards the products. Water had a strong effect on the activity of the enzyme and on K m . The concentration dependence of K m values was explained by the activity coefficients of the reacting agents. These were predicted with the thermodynamic models Perturbed-Chain Statistical Associating Fluid Theory (PC-SAFT), NRTL, and Universal Quasichemical Functional Group Activity Coefficients (UNIFAC), yielding the ratio of activity coefficients of products and reactants K γ . All model parameters were taken from literature. The models yielded K γ values between 25 and 115. Thus, activity coefficients have a huge impact on the consistent determination of the thermodynamic equilibrium constants K th. Combining K m and PC-SAFT-predicted K γ allowed determining K th and the standard Gibbs energy of reaction as function of temperature. This value was shown to be in very good agreement with results obtained from group contribution methods for Gibbs energy of formation. In contrast, inconsistencies were observed for K th using K γ values from the classical gE-models UNIFAC and NRTL. The importance of activity coefficients opens the door for an optimized reaction setup for enzymatic esterifications.

Thermodynamics of enzyme-catalyzed esterifications: II. Levulinic acid esterification with short-chain alcohols.

Levulinic acid was esterified with methanol, ethanol, and 1-butanol with the final goal to predict the maximum yield of these equilibrium-limited reactions as function of medium composition. In a first step, standard reaction data (standard Gibbs energy of reaction Δ R g 0 ) were determined from experimental formation properties. Unexpectedly, these Δ R g 0 values strongly deviated from data obtained with classical group contribution methods that are typically used if experimental standard data is not available. In a second step, reaction equilibrium concentrations obtained from esterification catalyzed by Novozym 435 at 323.15 K were measured, and the corresponding activity coefficients of the reacting agents were predicted with perturbed-chain statistical associating fluid theory (PC-SAFT). The so-obtained thermodynamic activities were used to determine Δ R g 0 at 323.15 K. These results could be used to cross-validate Δ R g 0 from experimental formation data. In a third step, reaction-equilibrium experiments showed that equilibrium position of the reactions under consideration depends strongly on the concentration of water and on the ratio of levulinic acid: alcohol in the initial reaction mixtures. The maximum yield of the esters was calculated using Δ R g 0 data from this work and activity coefficients of the reacting agents predicted with PC-SAFT for varying feed composition of the reaction mixtures. The use of the new Δ R g 0 data combined with PC-SAFT allowed good agreement to the measured yields, while predictions based on Δ R g 0 values obtained with group contribution methods showed high deviations to experimental yields.