Enhanced Lipase – Catalyzed Triglyceride Hydrolysis

The use of microbial lipases for the hydrolysis of natural oils such as triglyceride esters is a green alternative to conventional high temperature, high pressure steam-based technologies and other chemical synthesis. The hydrolytic splitting of the esters is necessary for the downstream production of high value chemical products such as coatings, adhesives, and high-performance personal care products. Deployment of enzymatic methods enables conversions to be achieved at close to ambient temperature and pressure with positive impacts on energy utilization and product purity. Enzymatic splitting of triglyceride esters is limited often by slow kinetics due to mass transfer limitations and by challenges of economic enzyme recycling. Immobilization of lipases on a solid carrier or support has proven to be an effective alternative method and, in some cases, considered superior to the use of lipases in aqueous media. Enhanced thermal and chemical stability, activity, recoverability, and reusability of the biocatalyst (lipase) are all potential advantages of immobilization. Efficacy of immobilization depends significantly on the mechanical integrity of the support, availability, and adjustable characteristics such as porosity, surface area, particle size a functional groups present, and the type of lipase used. The performance of microbial lipase derived from Candida rugosa immobilized onto polymeric methacrylate-based resins with no surface functionalization (ECR1030M), epoxy & butyl functionalization (ECR8285) and octadecyl functionalization (ECR8806M) supplied by Purolite® LifetechTM Resins Corporation was studied for triglyceride hydrolysis. The main mechanism involved in lipase immobilization was adsorption using hydrophobic interactions. Sizing of protein using the dynamic light scattering technique suggested immobilization was surface dominant. The resins were characterized using FT-IR spectrometry, N2 adsorption, contact angle measurements and scanning electron microscopy. Continuously stirred batch reactors operated at 100 RPM were used to compare the performance of the immobilized lipase by measuring the release of free fatty acids (FFA). Octadecyl functionalized methacrylate polymer resins showed superior performance. Comparing the octadecyl functionalized resins with un-functionalized methacrylate resins, a four-fold increase in activity retention was observed in multicycle experiments. Epoxy & butyl functionalized resins showed lower performance compared to octadecyl functionalized resin but higher than un-functionalized methacrylate resins. Performance in the presence of crosslinking agents such as, Glutaraldehyde, (3-Aminopropyl) triethoxysilane and itaconic acid applied during the immobilization protocol for methacrylate based polymeric resins and superior performance was observed in the case of itaconic acid on functionalized methacrylate resins. Prior work has shown that mass transfer rates and reaction rates can be intensified by increasing interfacial area for lipase catalyzed triglyceride hydrolysis using electrostatic spray reactors. However, the possible effect of oriented external electrical field on the lipase catalytic activity has hitherto not been considered. The performance of microbial lipase derived from Candida rugosa in aqueous solutions was analyzed in the presence of a steady DC externally applied electrical voltage. The reaction was conducted in three different batch type reactors: (1) In a quiescent (fixed interface) reactor; (2) In a stirred tank batch reactor, and (3) In a recirculating tubular flow reactor. It was concluded that the oriented external electrical field has a positive effect on all three-reactor system studied, showing reaction rate enhancement, independent of interfacial area. Further studies conducted using reverse polarity, increased electrode distance and for immobilized lipase system has shown that lipase undergoing conformational changes due to an oriented external electrical field is the main driving mechanism for this noted enhanced performance

Intensification of Liquid-Liquid Contacting Processes

In order to improve mass transfer rate and efficiently employ energy in liquid-liquid contacting processes, especially in biodiesel production through transesterification, two process intensification technologies were addressed based on two different energy sources and were investigated in this dissertation. They include electrostatic liquid spraying based on electric field and a two-disc spinning disc reactor based on high gravity field. Interfacial turbulence plays an important role in electrically enhanced mass transfer. One specific aspect of this work was to investigate interfacial turbulence which can occur at the interface between two phases because of interfacial tension gradients resulting from the change of charge density across droplet surface. Firstly, a Schlieren optical technique was developed and the experimental setup was built for the visualization of electrically induced interfacial phenomena in the ethanol-water-1-decanol system. Two Schlieren cells were designed and fabricated, and were successfully used for the interrogation of interfacial disturbances in pendant droplets and at plane interfaces. The mechanism of interfacial turbulence was further understood by study of these Schlieren images. Additionally, interfacial mass transfer in the ethanol-water-1-decanol system was investigated in the presence of electric fields and mass transfer coefficients were measured from the pendant droplets. Initial results show that a time-dependent nature was presented and mass transfer was intensified by the application of electric fields. As an alternative biofuel, biodiesel is produced through transesterification of vegetable oils, fat and algae lipids and alcohol with the help of acid or base. Transesterification is a liquid-liquid two phase reaction, whose rate is limited by mass transfer between oil and alcohol due to their immiscibility. This work firstly applied electrostatic liquid-liquid contactors to improve biodiesel synthesis. The reaction rate of transesterification of canola oil with sodium methoxide was investigated in the plane interface contactor. A fourfold enhancement was observed at an applied voltage of 10kV DC. A compact electrostatic spraying reactor based on simple tubular geometry with horizontal injection of an electrostatic spray of sodium methoxide was developed to achieve continuous biodiesel production. Preliminary data demonstrate it is a promising technology. A novel two-disc spinning disc reactor was developed as another alternative for biodiesel synthesis. It comprises two flat discs, located coaxially and parallel to each other with a very small gap between the discs. A grooved rotating disc was integrated to increase residence time allowing relatively slow transesterification reaction to be as complete as possible. The reaction performance was investigated widely. The conversion achieved in the reactor was significantly influenced by the size of inter-disc gap, the rotational speed, the canola oil phase flowrate, the surface topography of the rotating disc, and the reaction temperature. The experimental conditions were optimized. Finally, the new reactor was also compared with the stirred tank reactor according to the data of conversion rates and droplet size distribution of the emulsions

Process Intensification in a Liquid Biphasic Reaction System by Application of an External Electric Field

Due to the increasing concerns on global warming and depletion of fossil fuels, sustainable chemistry and engineering has been a research focus in the 21st century. Tremendous efforts have been devoted to designing inherently safe chemicals and processes with minimal waste emission and energy consumption. Among the many ongoing technologies, conducting chemistry in liquid biphasic systems is a promising approach and has found many successful applications in organic synthesis, biocatalysis, biomass pretreatment, etc. The development of green solvents, such as ionic liquids, further extends the scope of liquid biphasic systems for investigation of sustainable chemistry. While liquid biphasic systems feature many advantages, reactions in those systems usually suffer from mass transfer limitation. To facilitate mass transfer across the interfacial boundary, conventional methods, such as vigorous mechanical agitation or organic additions (e.g., phase transfer catalysts, or surfactants) are being extensively used. These methods, however, require significant energy input, increase the complexity of the reaction systems, and add to environmental concerns. To address this challenge, an intensification method achieved by application of external electric fields is proposed in this thesis. The electrostatic intensification in reaction performance in a liquid biphasic system was studied in two aspects: 1) controlling the migration of reactive species in a batch system, 2) increasing interfacial areas and reaction rates by application of an electrospray in a continuous system. This approach is easy to apply with relatively low energy consumption, thus showing great potential for a wide range of applications. Chapter I is an introduction of the research background. It discussed the importance and challenges of liquid biphasic systems in the development of sustainable chemistry and engineering. Three typical applications of liquid biphasic reaction systems, phase transfer catalysis, aqueous biphasic systems, and biomass processing, were discussed in terms of their abilities to achieve sustainability in chemistry and engineering. Recent advances in intensification methods in liquid biphasic systems were discussed with continuous flow processes, including microfluidic systems, and application of external electric fields as representatives. Afterwards, the model reaction system was discussed, followed by the proposals of research motivations and goals. In Chapter II, the roles of external electric fields in the batch reaction system were examined. It was demonstrated that water participated in the transfer hydrogenation of acetophenone when aqueous sodium formate was employed as hydrogen source. The external electric field was found to act as promoter or inhibitor for the phase transfer hydrogenation depending on the orientation of the electric field, which suggests the great potential of external electric fields in controlling reaction rates. In Chapter III, the ability of external electric fields to control reaction rates was further explored. It was found that the reaction performance was not linearly dependent on the applied voltages when the reaction time increased. The application of a negative voltage may result in the decomposition of the catalyst, which led to the decreased product enantioselectivity. It was also demonstrated that the reaction could be externally controlled by simply switching the applied electric potential over the course of the reaction. In Chapter IV, a continuous reaction system based on electrospray was established to further intensify mass transfer and reaction rates. Unlike the observations in Chapter II and III, the orientation of external electric fields was found to show no effects on the reaction performance. The induced electric current in the reaction system due to the increased conductivity of the continuous phase was proposed correlated to the improved conversion and yields. In this electrospray system, 71% conversion could be achieved in 1 h with a 13-fold increase in throughput compared to that in the batch system studied in Chapter II and III. In Chapter V, the importance of this work in understanding the roles of external electric fields in organic synthesis and catalysis was summarized. Further research directions and some new ideas were also proposed as complement to this thesis

BioMEMS

As technological advancements widen the scope of applications for biomicroelectromechanical systems (BioMEMS or biomicrosystems), the field continues to have an impact on many aspects of life science operations and functionalities. Because BioMEMS research and development require the input of experts who use different technical languages and come from varying disciplines and backgrounds, scientists and students can avoid potential difficulties in communication and understanding only if they possess a skill set and understanding that enables them to work at the interface of engineering and biosciences. Keeping this duality in mind throughout, BioMEMS: Science and Engineering Perspectives supports and expedites the multidisciplinary learning involved in the development of biomicrosystems. Divided into nine chapters, it starts with a balanced introduction of biological, engineering, application, and commercialization aspects of the field. With a focus on molecules of biological interest, the book explores the building blocks of cells and viruses, as well as molecules that form the self-assembled monolayers (SAMs), linkers, and hydrogels used for making different surfaces biocompatible through functionalization. The book also discusses: Different materials and platforms used to develop biomicrosystems Various biological entities and pathogens (in ascending order of complexity) The multidisciplinary aspects of engineering bioactive surfaces Engineering perspectives, including methods of manufacturing bioactive surfaces and devices Microfluidics modeling and experimentation Device level implementation of BioMEMS concepts for different applications. Because BioMEMS is an application-driven field, the book also highlights the concepts of lab-on-a-chip (LOC) and micro total analysis system (μTAS), along with their pertinence to the emerging point-of-care (POC) and point-of-need (PON) applications

Algae Biorefinery – Material and energy use of algae

Algae offer as much as 30 times greater biomass productivity than terrestrial plants, and are able to fix carbon and convert it into a number of interesting products. The numerous challenges in algae production and use extend across the entire process chain. They include the selection of suitable algal phyla, cultivation (which takes place either in open ponds or in closed systems), extraction of the biomass from the suspension, through to optimal use of the obtained biomass. The basic suitability of aquatic biomass for material use and energy supply has been demonstrated in a large number of studies. Numerous research projects are concerned with identifying the optimal processes to enable its widespread implementation. [... aus der Einleitung

Contact Mechanics and Adhesion of Polymeric Soft Matter Particles in Aqueous Environment

In the framework of this thesis, a study was conducted dealing with a strategy to change the mechanical properties of microgel particles using inwards-interweaving self-assembly post-synthesis. This technique was invented by my cooperation partners of the Trau group, and they prepared all particles studied. Exemplarily, agarose microparticles were used to interweave a defined shell of complexed poly(allylamine) (PA) and poly(styrenesulfonic acid) (PSS) into such particles. Thereby, the shell thickness can be well-defined. Adjusting the concentration of PA and the incubation time, the filling of the particles can be readily controlled up to complete filling. By adding excessive PSS, the diffusion-controlled shell formation stops by complexation. The shell thickness of individual particles was determined through fluorescence-labeled PA and confocal laser scanning microscopy. The mechanical properties of single particles were inferred by AFM with an attached colloidal probe (CP). Here, a non-linear increase of the elastic modulus (E-modulus) from 10 to 190 kPa was determined while the shell thickness increased from 10 to 24 µm. After adding a second shell, a further gain to 520 kPa on average can be realized. Furthermore, a new concept was developed by Mr. Fery and me to change the surface mechanical properties of mircogel particles by applying a thermal trigger. Meanwhile, a particular focus was laid to maintain constant adhesive properties at every temperature. First, crosslinked poly(N-isopropyl acrylamide) (PNIPAM) particles are prepared by droplet microfluidics. These gelled particles are injected into a second microfluidic device and surrounded by an aqueous solution of uncrosslinked poly(acrylamide) (PAAM). At a second junction, droplets are formed via the cut-off effect of the continuous organic solvent. The droplets now contain the crosslinked PNIPAM core and a thin uncrosslinked PAAM liquid shell. After a short diffusion of PAAM polymers into the core they are crosslinked by UV-light. These experiments were performed by our cooperation partners of the Seiffert group. I applied temperature-controlled CP-AFM to obtain the resulting adhesive and mechanical properties of individual particles. Here, the core-shell particles behaved similarly to plain PNIPAM particles displaying the typical increase in E-modulus at temperatures above 34°C, however lower in magnitude. Further, no temperature effect on the interfacial interaction for these core-shell particles was detected. While one focus of the study above was on constant adhesion, in the following section, a new synthetic approach for mussel inspired underwater adhesives, and their characterization is presented. Based on a peptide sequence of ten amino acids, which is found frequently in natural mussel foot proteins, a new polymerization route was developed by my cooperation partners of the Börner group. Possible reaction pathways were investigated with specifically designed model reaction and analyzed using mass spectroscopy and gel permeation chromatography. The resulting polymers were further characterized using high-performance liquid chromatography and SDS-page. Nanometer thick coatings of these synthetic polymers revealed an excellent persistence even against highly concentrated salt solutions measured by quartz crystal microbalance with dissipation experiments. My contribution was the investigation of the work of adhesion necessary to detach a microparticle from such a coating by CP-AFM in an aqueous environment. Here, the newly developed synthetic polymer provided higher adhesive strength, up to 10.9 mJ m- 2, compared to comparable natural mussel foot proteins.Im Rahmen dieser Arbeit, wurde eine Studie durchgeführt, welche die Möglichkei¬ten der nachträglichen Elastizitätsveränderung von Mikrogelpartikeln mittels „nach Innen gerichteter, verwebender Selbstassemblierung“ (engl. inwards-interweaving self-as¬sembly) beleuchtet. Diese Technik wurde von meinen Kooperationspartnern aus der Gruppe von Herrn Trau entwickelt. Am Bespiel von Agarose Mikropartikeln, kann mittels dieser Technik eine definierte Schale aus Polyallylamin (PA) und Polystyrolsulfonsäure (PSS) in das Partikel verwoben werden. Die Schalendicke kann dabei kontrolliert variiert werden, bis hin zur vollständigen Ausfüllung des Partikels, in dem die Konzentration von PA und die Inkubationszeit angepasst werden. Durch Zugabe eines Überschusses an PSS wird der diffusionsgesteuerte Schalenaufbau durch Komplexierung beendet. Die Schalendicke der individuellen Partikel wurde mittels Fluoreszenzmarkierung und konfokaler Laser Raster¬mikroskopie (engl. confocal laser scanning microscopy) ermittelt. Die mechanische Cha¬rakterisierung einzelner Partikel durch AFM und kolloidaler Sonde (engl. colloidal probe, CP) ergab eine nicht lineare Erhöhung des Elastizitätsmoduls (E-Modul) von 10 auf 190 kPa bei einem Schalendicken Zuwachs von 10 auf 24 µm. Durch eine zweite Schale, in der Ersten, konnte der E-Modul auf im Mittel 520 kPa gesteigert werden. Weiterführend, wurde von mir und Herrn Fery ein neues Konzept entwickelt, um eine mechanische, oberflächliche Verhärtung von Mikrogelpartikel durch Temperaturver¬änderung zu induzieren mit einem Augenmerk, dass sich die Adhäsionseigenschaften nicht verändern. Zunächst wurden von meinen Kooperationspartnern aus der Gruppe von Herrn Seiffert vernetzte Poly-N-isopropylacrylamid (PNIPAM) Partikel mittels Tropfenmikroflu¬idik hergestellt. In einem zweiten Mikrofluidik Experiment wurde diese Partikel mit einer wässrigen Lösung von Polyacrylamid (PAAM, unvernetzt) umgeben bevor es zu einer Tropfenbildung in der organischen Phase kommt. Nach kurzer Diffusionszeit der PAAM Polymerketten in die Kernpartikel, wurde die PAAM Schale mittels UV-Licht querver-netzt. In temperaturkontrollierten CP-AFM Untersuchungen habe ich die resultierenden Adhäsions- und mechanischen Eigenschaften auf der Einzelpartikel Ebene bestimmt. Hier¬bei konnte bei den Kern-Schale Partikeln der für bloße PNIPAM Partikel typische E-Modul anstieg oberhalb von 34°C nachgewiesen werden, jedoch mit verminderten Absolutwerten. Eine begleitende Veränderung der adhäsiven Eigenschaften der Kern-Schale Partikel konnte dabei nicht beobachtet werden. Lag ein Fokus der vorherigen Arbeit auf konstanten Wechselwirkungen, behandelt der dritte Teil der Ergebnisse, einen neuen Synthese Ansatz zur Herstellung Muschel in¬spirierter Unterwasser Adhäsiva und deren Charakterisierung. Basierend auf einer natürli¬chen Peptidsequenz, wurde eine enzymatische Polymerisationsroute von meinen Koopera¬tionspartner aus der Börner Gruppe entwickelt. Der Reaktionsverlauf wurde durch neu de¬signte Modelreaktion untersucht und mittels Massenspektroskopie und GPC analysiert, das resultierende Polymer zusätzlich mit HPLC und SDS-page. Nanometer dicke Beschichtun¬gen dieser Muschel inspirierten Polymer wiesen eine sehr gute Beständigkeit gegen hoch konzentrierten Salzlösung in QCM-D Experimenten auf. Die Adhäsionsarbeit, welche nö¬tig ist um eine Mikropartikel von diesen in wässeriger Lösung zu entfernen, wurde von mir mittels CP-AFM bestimmt. Nach meiner Erweiterung einer bekannten Adhäsionstheorie, konnten für das synthetische Polymer höhere Werte als für vergleichbare Natürliche von bis zu 10.9 mJ m-2 bestimmt werden