MSc in Biotechnology
Proposal for Research Project – 2nd Cycle
Tema transitado dos não escolhidos do ano passado. A aluna iniciou dissertação
no segundo semestre. Aprovado CC
Student´s Name: Cláudia Mendes da Costa
Student email address:
No. 45887
Supervisor: Susana Barreiros
Supervisor´s email address: sfb@fct.unl.pt
Co-supervisor: João Paulo Borges
Co-cupervisor’s email address: jpb@fct.unl.pt
Lab/Institution: Lab 427 DQ-REQUIMTE and Lab 211 DCM-CENIMAT/i3N
Scientific area: Industrial Biotechnology
TITLE: Biocatalytic fiber membrane for the valorization of CO2 into a biofuel.
BACKGROUND
Carbon dioxide utilization is seen as a complementary approach to carbon capture and storage. The
conversion of CO2 into useful products, such as methanol, a primary liquid petrochemical of
considerable relevance in the chemical and energy industries, becomes increasingly important as CO2
levels in the atmosphere continue to rise. The conversion of CO2 to methanol can be accomplished by
three dehydrogenases – formate (FateDH), formaldehyde (FaldDH) and alcohol (ADH) dehydrogenase –
working in sequence, requiring the action of the cofactor reduced nicotinamide adenine dinucleotide
(NADH).
Through electrospinning it is possible to obtain easily micro- and nanofibers with high superficial area
and controlled porosity, through the action of electrical fields. Confinement within fibers confers
protection and stability to bioactive molecules and facilitates molecular interactions.
Core/shell structured fibers can be designed so as to combine the versatility of a microenvironment
that suits the action of the biomolecules, with an outer layer that suits the medium and application
envisaged for the biocatalytic system.
OBJECTIVES
The objective of the workplan is to fabricate, using coaxial electrospinning, reactive fibers containing a
biocatalytic system able to reduce CO2 to methanol, comprising a biocompatible core entrapping
enzymes and co-factor, and a silica-based, mechanically resistant, porous shell.
PROJECT DESCRIPTION
Task 1 - Fabrication of a core/shell structured fiber membrane – Months 01-08
The biocatalytic process involves ion transfer. In the system envisaged there will not be a bulk water
phase. Therefore the fiber cores will be produced with an ionic gel, a conductive polymer composed of
an ionic liquid(IL) and gelatin that provides a microenvironment able to solvate charged species. Ionic
gels have been shown to provide an adequate medium for immobilizing enzymes belonging to the
same class of the dehydrogenases.
Based on previous experience in the fabrication of IL/gelatin electrospun fibers with choline-based ILs,
a biocompatible, hydrated, choline dihydrogen phosphate(dhp)/monohydrogen phosphate(hp), with a
buffering action at a pH that is convenient for the biocatalysts will be used.
The choice of silica to fabricate fiber shells is based on its low cost, manufacturability, mechanical,1
thermal and pH stability, and chemical inertness. Tetramethoxysilane (TMOS) or tetraethoxysilane
(TEOS), the silica precursors most commonly used, will be used here too. To overcome the brittleness
of silica, the silica precursor will be mixed with a biocompatible polymer (polyvinyl alcohol, PVA, or
polyvinyl acetate, PVAc). This will also allow the tailoring of the porosity of the shell.
MSc in Biotechnology
Proposal for Research Project – 2nd Cycle
A coaxial electrospinning setup coupled with a microfluidic timer will be used (Figure 1). The system
will be first tested without catalysts. The core solution, containing IL, gelatin and water will be loaded
into syringe A, a silica sol-gel precursor solution into syringe B, and a PVA or PVAc solution into syringe
C. The microfluidic timer will ensure that an optimum viscosity of the mixture of hydrolyzed sol-gel
precursor and polymer solutions is reached during gelation before electrospinning.
The optimization of the electrospinning process will be done taking into account environmental
parameters (temperature and humidity), solution properties (viscosity, conductivity, surface tension),
and the process itself (applied voltage, capillary-collector distance, solution flow rate). The effects of
critical design parameters - flow rates of the solutions (core and shell), protrusion of the core needle,
viscosity of the solutions, on the reproducibility and quality of the fibers will be assessed.
Different gelatin and PVA(or PVAc) concentrations will be used to produce the core and shell solutions,
respectively.
The fibers will be characterized in terms of morphology, using Scanning Electron Microscopy. Other
techniques available may be used, if time allows, to determine surface area (Brunauer-Emmett-Teller
analysis), pore size (mercury porosimetry), physico-chemical properties (Infrared spectroscopy) and
mechanical properties (tensile tests).
Task 2 - Biocatalytic system – Months 03-08.
a) First, only the last enzyme in the biocatalytic sequence will be used, namely ADH, the least
expensive one. In the envisaged process, it will convert formaldehyde to methanol. Here, it will be
used to function as it normally does, i.e. in reverse. In the present work, ADH will be used to convert
methanol to formaldehyde, with the help of cofactor NAD+. The enzyme will be studied in choline
dhp/hp medium with different water contents, determined by Karl-Fischer titration. The effect of the
water content of the IL medium on cofactor solubility will be assessed. The formation of NADH will be
monitored by UV-VIS spectroscopy at λ = 340 nm.
b) The three-enzyme system will be assayed in a few experiments carried out at conditions set by
reactions done in a), using NAD+ as co-factor to convert methanol to CO2.
c) After performing assays in IL medium, ADH will be immobilized in the IL/gelatin ionic gel. Enzymatic
reactions will be carried out in an apolar organic solvent unable to strip water off the system.
Reactions will be followed by gas chromatography (GC).
d) After conducting these assays, the three enzymes will be co-immobilized together with NAD+ in
ionic gels, and their performance will be tested in a few experiments performed at optimized
conditions.
e) Finally, the bioconversion of CO2 to methanol using NADH as cofactor will be assayed. Experiments
will be conducted in a cell with CO2 at high pressure. Reaction progress will be monitored by taking
samples and quantifying methanol by GC.
Task 3 – Biocatalytic fiber membrane reactor – Months 09-10.
After gaining experience in producing blank, low-defect fibers, the procedures developed in Task 1 will
be extended to the fabrication of core/shell nanofibers holding the biocatalytic system.
As a proof of concept, the mat of fibers will be placed inside the high pressure cell. Reactions will be
carried out with high pressure CO2, and methanol formation will be followed by GC.
Task 4 – Writing of dissertation thesis – Months 11-12.
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MSc in Biotechnology
Proposal for Research Project – 2nd Cycle
Figure 1 - Electrospinning setup for producing core/shell nanofibers with an ionic gel core and a silicabased shell. The system is similar to that used by Tong et al.[1]. (a) Basically, it utilizes a coaxial
electrospinning setup (syringe A) coupled with a microfluidic timer (connecting syringes B and C); (b) The
chemical reaction between the hydrolyzed sol-gel precursor solution (syringe B) and the polymer solution
(syringe C) during the electrospinning process is controlled by the microfluidic timer.
[1] H.-W. Tong, B.R. Mutlu, L.P. Wackett, A. Aksan, Manufacturing of bioreactive nanofibers for
bioremediation, Biotechnol. Bioeng. 111 (2014) 1483–93.
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