2014 MS Thesis topics HES-SO Valais Wallis, Biotechnology Unit Prof. Simon Crelier

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1 2014 MS Thesis topics HES-SO Valais Wallis, Biotechnology Unit Prof. Simon Crelier A. Lab s activities Hosted in the Life Technologies building of the HES-SO Valais Wallis in Sion, the laboratory is active in various areas such as the isolation of biomolecules issued from extracts or fermentations processes, the technology, matrices and applications of microencapsulation, process intensification as well as enzyme technology at large. Our research efforts feature academic projects as well as industrial collaborations, and our developments usually are of an applied nature. Various topics are described in the next section B. Summarized list 1. Improved design for a bench-scale expanded bed adsorption equipment 2. In situ measurement of adsorption kinetics 3. Use of microfluidic devices for bioseparations 4. Scaling-up of an enzyme purification using ATPS 5. Enzymatic microreactors 6. Biotransformations using immobilized enzymes 7. Biosorption for the removal of micropollutants 8. Production and isolation of cellulases 9. Saccharification of waste paper using industrial enzymes 10. Biogas production optimization of an anaerobic digestor C. Description 1 Improved design for a bench-scale expanded bed adsorption equipment Expanded bed Adsorption (EBA) is an interesting development for the downstream processing of biomolecules; its main advantage is the adsorption of the target compound directly from a non-clarified cell suspension or lysate. Turbid material containing cell debris and/or precipitated material can be injected in a bed of suspended particles with no risk of plugging the column. However, the performance of that kind of installation can be severely impaired by the following aspects: 1

2 Imperfect verticality of the column, pump pulsation. Inhomogeneous distribution of the liquid at the column inlet, which leads to channelling and shallow breakthrough curves. Height of the void space between fluid bed expansion front and liquid collection point. The larger the headspace, the more important the backmixing and the broadening of the peak. Since expansion increases with both liquid viscosity and density, this level fluctuates when one switches from fermentation broth to washing buffer and later to elution mixture. The main goal of this project is to propose improvements to existing lab-scale equipments, implement the proposed changes and characterize the impact of the column modifications on the performance of the installation. A model fermentation broth will be selected for the various stages of the investigation. 2 In situ measurement of adsorption kinetics In the development of chromatographic separations, the characterization of the matrix is a key step. Measurements of adsorption capacity and kinetics as well as selectivity are a necessary, but very time-consuming stage of the development which involve numerous samplings, dilutions and measurements. The goal of the present project is the development and validation of an improved, faster experimental protocol for the characterization of chromatographic adsorbents. In this approach a fibre optics probe will be dipped directly into the mixed suspension, hence allowing a direct measurement of the adsorption kinetics without any sampling of liquid. The main goal of the present approach is to compare the on-line measurements with the classical, off-line procedure and validate the former. Steps in this study will involve the selection of suitable and relevant experimental models comprising adsorbates (e.g. phenol, methylene blue, caffein, antibiotic) as well as adsorbents (active coal, ion exchange resin, biosorbent). The obtained kinetics and equilibrium data will, in a final step, be used to model fixed-bed adsorption experiments and the resulting breakthrough curves. 3 Use of microfluidic devices for bioseparations Microreactors, microfluidics components and their use in the field of process intensification represent a very exciting and continuously expanding field of investigation. However, little has been done so far to explore the potential of these devices in the field of downstream processing unit operations. Based on some examples from the literature, techniques such as 2

3 liquid-liquid extraction, aqueous two-phase extraction, microencapsulation and (nano)precipitation will be tested using different microchip configurations. The goal of this project is to explore different configurations of microfluidic chips and evaluate their potential for the design, optimization and scaling-up of model bioseparations. 4 Scaling-up of an enzyme purification using ATPS Aqueous Two Phase Systems are the subject of renewed attention for the purification of proteins out of a fermentation broth or cell lysate. This technique can handle large volumes at a relatively modest cost and represents an interesting alternative to more expensive approaches. In the course of this project, a suitable extraction system will be selected for the extraction of an enzyme, based on a first screening. The selected aqueous two-phase system will then be characterized at small scale with respect to its phase separation behaviour (measurement of the binodal curves and tie-lines) and partition coefficients of the target enzyme and contaminant proteins. The optimized extraction will then be conducted at two larger scales (1.0 L and 20.0 L) for comparison. A response surface methodology such as described in Zhi et al. (2005) shall be applied for the optimization. Zhi W., Song J., Ouyang F., Bi J. (2005). Application of response surface methodology to the modeling of α-amylase purification by aqueous two-phase systems. Journal of Biotechnology 118 (2), Enzymatic microreactors Microreaction engineering has developed tremendously in the last few years and has become a very exciting approach for the development and optimization of reaction parameters and processes under safe conditions. In the field of enzyme reaction it is the cost of reagents rather than the exothermicity which can be better controlled by working with small volumes and flow rates. Also, that configuration allows the rapid screening of multiple reaction conditions. Microchannels can also be tailor-made at a relatively low price in order to accommodate particles of immobilized catalysts such as enzymes. The main goal of this project is the construction and characterization of such an enzymatic microreactor. A model reaction will be studied and optimized with respect to mixing efficiency (a critical point at low Reynolds numbers), temperature, ph, yield, and potential for on-line analytics and up-scaling. 3

4 Comparison with more classical designs (fixed bed catalytic reactors) will also be part of the investigation. 6 Biotransformations using immobilized enzymes There are a large variety of methods for the immobilization of enzymes, but whatever the approach a negative impact on mass transfer is to be expected. Several immobilization protocols will be tested and their impact on enzyme activity and stability carefully assessed. The most efficient catalyst will be selected based on the outcome of this first step. The reaction kinetics will be characterized and compared with the soluble enzyme. Biotransformations using the enzyme shall then be performed in batch as well as in fixed bed configuration and characterized. The hydrolysis of penicillin G with penicillin G acylase (EC ) will be used as a model reaction. Remark: this subject can be combined with topic N 5 7 Biosorption for the removal of micropollutants Micropollutants have become a real environmental threat in the last few years, and ways of resolving this problem are being sought for actively. Finding alternative, greener chemistries is certainly the most efficient approach, but there is simultaneously a significant amount of research aiming at reducing their dissemination into the environment as well as selectively removing them from our rivers and lakes. Biosorption is one of these approaches, and consists in using low-cost, inert biomasses in order to adsorb certain compounds. Adsorption has the advantage of being most efficient at low concentrations. Large volumes of liquid can thus be processed and stripped from their contaminants, which gradually concentrate on the adsorbent. Once equilibrium has been reached the contaminant can be eluted in a concentrated form which allows either its costeffective destruction or its re-use. The goal of this work will be to investigate various biosorbents (with environmentally relevant micro contaminants), optimize their formulation/conditioning and characterize them with respect to adsorption kinetics and capacity. 8 Production and isolation of cellulases Cellulases are an indispensable tool for the degradation of cellulosic biomass and the production of bioethanol or biobased materials. They usually consist in a mixture of three major activities: endo-ß-glucanase (EC ), exo-ß-glucanase (EC ) and ß- glucosidase (EC ). 4

5 A first part of this project will consist in isolating cellulase-producing micro-organisms from the environment using screening techniques. The selected strains will be cultivated in bioreactors and the cellulase activities isolated. The latter will then be characterized in terms of molecular weight, isoelectric point, activity and stability. Finally, saccharification trials will be performed using various substrates. 9 Saccharification of waste paper using industrial enzymes The aim of this project is to test the efficiency of various commercial enzyme preparations for the saccharification of different sorts of waste paper. The substrates as well as the products will be characterized and optimal conditions of ph and temperature will be sought. The kinetics of the enzymatic degradation will then be measured and modelled. In a final phase of the project, the product of the saccharification step will be used in a fermentation to produce bioethanol. 10 Biogas production optimization of an anaerobic digestor Our laboratory is involved in a European project of the 7 th framework called ORION ( Its main objective is the development of a new generation of anaerobic digester operating at the SME scale (1 to 50 m 3 ) and thus allowing on-site treatment of the waste. Our task is to assess the digestibility of various substrates, mainly organic waste issued from agriculture, open air markets, food processing industry, restaurants or agriculture. The project will imply the analytical monitoring of the digestion process and its optimization with respect to organic loading rate, biogas production and reduction of the environmental burden. Contact person Prof. Simon Crelier HES-SO Valais Wallis Life Technologies / Biotechnology Route du Rawyl 64 CH-1950 Sion 2, Switzerland Tel. +41 (0) simon.crelier@hevs.ch 5