Mathias Nordblad

Wax esters produced by solvent-free energy-efficient enzymatic synthesis and their applicability as wood coatings

The study aimed at developing a process for making a wood coating wax based on the principles of green chemistry. The research was conducted within the Swedish interdisciplinary research programme Greenchem. Wax esters are attractive since they are non-hazardous, biodegradable and can be produced in an atom-efficient process from building blocks obtained from renewable resources. Four wax esters were prepared in a solvent-free process using an immobilised lipase as catalyst. When the water was removed during the process from what was initially an equimolar mixture of the starting materials carboxylic acid and alcohol by a stream of dry air passed through the reactor, there was a 95–99% conversion to the ester. The enzymatic process consumed 34% less energy and generated less waste than chemical esterification using a strong acid as catalyst. Two of the esters worked well in the industrial wood coating equipment employed and produced surfaces resistant to water and somewhat less to fat stains.

Identification of critical parameters in liquid enzyme-catalyzed biodiesel production

Callera™ Trans L, a liquid formulation of Thermomyces lanuginosus lipase, has recently shown great promise as a cost‐efficient catalyst for methanolysis of triglyceride substrates, specifically in the BioFAME process. However, identifying the right combination of temperature and concentrations of catalyst, water and methanol to realize the full potential of the reaction system has remained a challenge. This study presents an investigation of the impact of temperature, enzyme and water concentration on the reaction, as well as the effect of methanol feed rate for the conversion of rapeseed oil in a fed‐batch reaction system. It was observed that the reaction can be divided into two distinct parts. The first part of the reaction, during which primarily tri‐ and diglycerides are converted, proceeded at a high rate and thus required a high rate of methanol supply. The second part of the reaction, where the remaining di‐ and monoglycerides are converted, proceeded at a much lower rate. Consequently, it is necessary to reduce the methanol feed rate during the latter part of the reaction to avoid inhibition or even inactivation of the enzyme. Since the second part of the reaction occupied most of the 24‐h reaction time, it was concluded that this is the part of the process where further development efforts should be targeted. This point was demonstrated by partially substituting the catalyst with a lipase with a different specificity, which enhanced the performance during the second phase of the reaction. Biotechnol. Bioeng. 2014;111: 2446–2453.

Chemo-enzymatic Epoxidation―Process Options for Improving Biocatalytic Productivity

The reactor choice is crucial when designing a process where inactivation of the biocatalyst is a problem. The main bottleneck for the chemo‐enzymatic epoxidation has been found to be enzyme inactivation by the hydrogen peroxide, H2O2, substrate. In the work reported here, the effect of reaction parameters on the reaction performance have been investigated and used to establish suitable operating strategies to minimize the inactivation of the enzyme, using rapeseed methyl ester (RME) as a substrate in a solvent‐free system. The use of a controlled fed‐batch reactor for maintaining H2O2 concentration at 1.5 M resulted in increased productivity, up to 76 grams of product per gram of biocatalyst with higher retention of enzyme activity. Further investigation included a multistage design that separated the enzymatic reaction and the saturation of the RME substrate with H2O2 into different vessels. This setup showed that the reaction rate as well as enzyme inactivation is strongly dependent on the H2O2 concentration. A 20‐fold improvement in enzymatic efficiency is required for reaching an economically feasible process. This will require a combination of enzyme modification and careful process design.

Mechanistic modeling of biodiesel production using a liquid lipase formulation

In this article, a kinetic model for the enzymatic transesterification of rapeseed oil with methanol using Callera™ Trans L (a liquid formulation of a modified Thermomyces lanuginosus lipase) was developed from first principles. We base the model formulation on a Ping‐Pong Bi‐Bi mechanism. Methanol inhibition, along with the interfacial and bulk concentrations of the enzyme was also modeled. The model was developed to describe the effect of different oil compositions, as well as different water, enzyme, and methanol concentrations, which are relevant conditions needed for process evaluation, with respect to the industrial production of biodiesel. The developed kinetic model, coupled with a mass balance of the system, was fitted to and validated on experimental results for the fed‐batch transesterification of rapeseed oil. The confidence intervals of the parameter estimates, along with the identifiability of the model parameters were presented. The predictive capability of the model was tested for a case using 0.5% (wt. Enzyme/wt. Oil), 0.5% (wt. Water /wt. Oil) and feeding 1.5 times the stoichiometric amount of methanol in total over 24 h. For this case, an optimized methanol feeding profile that constrains the amount of methanol in the reactor was computed and the predictions experimentally validated. Monte‐Carlo simulations were then used to characterize the effect of the parameter uncertainty on the model outputs, giving a biodiesel yield, based on the mass of oil, of 90.8 ± 0.55 mass %.

Scale-up of industrial biodiesel production to 40 m3 using a liquid lipase formulation

In this work, we demonstrate the scale‐up from an 80 L fed‐batch scale to 40 m3 along with the design of a 4 m3continuous process for enzymatic biodiesel production catalyzed by NS‐40116 (a liquid formulation of a modified Thermomyces lanuginosus lipase). Based on the analysis of actual pilot plant data for the transesterification of used cooking oil and brown grease, we propose a method applying first order integral analysis to fed‐batch data based on either the bound glycerol or free fatty acid content in the oil. This method greatly simplifies the modeling process and gives an indication of the effect of mixing at the various scales (80 L to 40 m3) along with the prediction of the residence time needed to reach a desired conversion in a CSTR. Suitable process metrics reflecting commercial performance such as the reaction time, enzyme efficiency, and reactor productivity were evaluated for both the fed‐batch and CSTR cases. Given similar operating conditions, the CSTR operation on average, has a reaction time which is 1.3 times greater than the fed‐batch operation. We also showed how the process metrics can be used to quickly estimate the selling price of the enzyme. Assuming a biodiesel selling price of 0.6 USD/kg and a one‐time use of the enzyme (0.1% (w/woil) enzyme dosage); the enzyme can then be sold for 30 USD/kg which ensures that that the enzyme cost is not more than 5% of the biodiesel revenue. Biotechnol. Bioeng. 2016;113: 1719–1728.

Biocatalytic polyester acrylation--process optimization and enzyme stability.

An OH‐functional polyester has been acrylated via transesterification of ethyl acrylate, catalyzed by Candida antarctica lipase B (CalB) in two different preparations: Novozym® 435 and immobilized on Accurel® MP1000. The batch process resulted in incomplete acrylation as well as severe degradation of the polyester. A high degree of acrylation was achieved by optimization through the application of low pressure (15 kPa), continuous inflow of ethyl acrylate and continuous distillation to evaporate the by‐product, ethanol. The enzyme preparations displayed good stability with half‐lives of 180 and 324 h for Novozym® 435 and CalB/MP1000, respectively. This translates into product yields of 3600 and 6200 times the weight of the catalyst, indicating that the enzyme will have a marginal impact on the total process cost. Biotechnol. Bioeng. 2009; 102: 693–699.

Immobilisation procedure and reaction conditions for optimal performance of Candida antarctica lipase B in transesterification and hydrolysis

Abstract The reaction kinetics of Candida antarctica lipase B (CalB) in the commercially available preparation Novozym® 435 (N435) were compared to those of preparations of CalB immobilised on Accurel® MP1000 (porous polypropylene). Two polypropylene preparations were made using enzyme loadings of 0.2% and 2% (w/w). All three preparations were used in hydrolysis as well as transesterification of two substrates, ethyl acrylate and ethyl methacrylate with octanol. Reactions carried out at water activity levels from 0.06 to 0.96 and at octanol concentrations between 25 and 500 mM showed that both water and octanol can inhibit CalB. Pronounced mass transfer limitations were also observed, which were more pronounced for N435 than for the two MP1000 preparations. The MP1000 preparations could thus use the lipase more efficiently in these reactions, achieving a specific activity (per g enzyme) between 5 and 20 times that of N435. To achieve high rates in the transesterification reaction, it is recommended to use low water activity and moderate alcohol concentration. In order to carry out a hydrolysis reaction, an intermediate water activity should be used to balance the effects of water as a limiting substrate and as a competitive inhibitor.

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.

Real‐time model based process monitoring of enzymatic biodiesel production

In this contribution we extend our modelling work on the enzymatic production of biodiesel where we demonstrate the application of a Continuous‐Discrete Extended Kalman Filter (a state estimator). The state estimator is used to correct for mismatch between the process data and the process model for Fed‐batch production of biodiesel. For the three process runs investigated, using a single tuning parameter, qx = 2 × 10−2 which represents the uncertainty in the process model, it was possible over the entire course of the reaction to reduce the overall mean and standard deviation of the error between the model and the process data for all of the five measured components (triglycerides, diglycerides, monoglycerides, fatty acid methyl esters, and free fatty acid). The most significant reduction for the three process runs, were for the monoglyceride and free fatty acid concentration. For those components, there was over a ten‐fold decrease in the overall mean error for the state estimator prediction compared with the predictions from the pure model simulations. It is also shown that the state estimator can be used as a tool for detection of outliers in the measurement data. For the enzymatic biodiesel process, given the infrequent and sometimes uncertain measurements obtained we see the use of the Continuous‐Discrete Extended Kalman Filter as a viable tool for real time process monitoring.

Online Measurement of Oxygen‐Dependent Enzyme Reaction Kinetics

As the application of biocatalysis to complement conventional chemical and catalytic approaches continues to expand, an increasing number of reactions involve poorly water‐soluble substrates. At required industrial concentrations necessary for industrial implementation, this frequently leads to heterogeneous reaction mixtures composed of multiple phases. Such systems are challenging to sample, and therefore, it is problematic to measure representative component concentrations. Herein, an online method for following the progress of oxygen‐dependent reactions through accurate measurement of the oxygen mass balance in the gas phase of a reactor is demonstrated and validated. The method was successfully validated and demonstrated by using two model reactions: firstly, the oxidation of glucose by glucose oxidase and, secondly, the Baeyer–Villiger oxidation of macrocyclic ketones to lactones. Initial reaction rate constants and time‐course progressions calculated from the oxygen mass balance were validated against conventional online methods of dissolved oxygen tension and pH titration measurements. A feasible operating window and the sensitivity to dynamic changes of reaction rates were established by controlling oxygen transfer through the operating parameters of the reactor. Such kinetic data forms the basis for reaction characterisation, from which bottlenecks may be made evident and directed improvement strategies can be identified and implemented.