Rosalinda Mazzei

Purification of triacylglycerols for biodiesel production from Nannochloropsis microalgae by membrane technology

Triacylglycerols recovery from wet microalgae is a key aspect of biodiesel production, because of the energetic balance gained from avoiding biomass drying. In order to isolate TAG from Nannochloropsis cells, the possibility to concentrate biomass and to recover TAG in a single step by membrane process was studied. Different polymeric membranes were selected and screened on the basis of adsorption test and permeation flux. Results showed that membrane of regenerated cellulose (RC) with nominal molecular weight cutoff of 100 kDa and 30 kDa gave the best performance. Indeed, permeate flux was stable during ultrafiltration experiment in concentration mode and no severe fouling/cake deposition was observed. Both membranes allowed to recover permeates with high content of triacylglicerols. However, a more purity of the triacylglicerols from the other co-products was only obtained with the 30 kDa RC membrane because the retention of the unwanted proteins was in the range of 89%.

Influence of protein bulk properties on membrane surface coverage during immobilization

Biomolecules immobilization is a key factor for many biotechnological applications. For this purpose, the covalent immobilization of bovine serum albumin (BSA), lipase from Candida rugosa and protein G on differently functionalized regenerated cellulose membranes was investigated. Dynamic light scattering and electrophoresis measurements carried out on biomolecules in solution indicated the presence of monomers, dimers and trimers for both BSA and protein G, while large aggregates were observed for lipase. The immobilization rate and the surface coverage on functionalized regenerated cellulose membranes were studied as a function of biomolecule concentration. Results indicated that the saturation coverage of BSA and protein G was concentration independent (immobilized protein amount of 2.40±0.03mg/g and 2.65±0.07mg/g, respectively). Otherwise, a different immobilization kinetics trend was obtained for lipase, for which the immobilized amount increases as a function of time without reaching a saturation value. Atomic force microscopy (AFM) micrographs showed the formation of monolayers for both BSA and protein G on the membrane surface, while a multilayer structure is found for lipase, in agreement with the trends observed in the related immobilization kinetics. As a result, the morphology of the proteins layer on the membrane surface seems to be strictly dependent on the proteins behavior in solution. Besides, the surface coverage has been described for BSA and protein G by the pseudo second order models, the results indicating the surface reaction as the controlling step of immobilization kinetics. Finally, enzyme activity and binding capacity studies indicated the preservation of the biomolecule functional properties.

Pectinases immobilization on magnetic nanoparticles and their anti-fouling performance in a biocatalytic membrane reactor

Enzyme immobilization on commercial superparamagnetic nanoparticles (NPSP) was performed using covalent bonding. The biofunctionalized NPSP was then immobilized on the surface of the membrane using an external magnetic field to form a magneto-responsive biocatalytic membrane reactor (BMRSP). The magnetically formed smart nanolayer can be easily re-dispersed and recovered from the membrane when the enzyme is deactivated or whenever cleaning is required due to substrate over-accumulation. The system was used to hydrolyze pectin contained in different streams. Results are supported with complementary data from hydrodynamic, kinetic and morphological characterization in a flow-through reactive filtration. Wavelength-dispersive X-ray spectroscopy (WDS) elemental mapping revealed that the NPSP are uniformly dispersed on the surface of the membrane forming a thin biocatalytic layer. Both results of hydrodynamic studies and SEM micrographs of the membrane with the enzyme layer under various operating conditions, show that the immobilized enzyme effectively reduced membrane–foulant interaction. Comparison of filtration data using this commercial NPSP reveals good agreement with our previously used home-made NPSP. This implies that the scaling-up and commercialization of the developed BMRSP can be straightforward.

Use of a Ceramic Membrane to Improve the Performance of Two-Separate-Phase Biocatalytic Membrane Reactor

Biocatalytic membrane reactors (BMR) combining reaction and separation within the same unit have many advantages over conventional reactor designs. Ceramic membranes are an attractive alternative to polymeric membranes in membrane biotechnology due to their high chemical, thermal and mechanical resistance. Another important use is their potential application in a biphasic membrane system, where support solvent resistance is highly needed. In this work, the preparation of asymmetric ceramic hollow fibre membranes and their use in a two-separate-phase biocatalytic membrane reactor will be described. The asymmetric ceramic hollow fibre membranes were prepared using a combined phase inversion and sintering technique. The prepared fibres were then used as support for lipase covalent immobilization in order to develop a two-separate-phase biocatalytic membrane reactor. A functionalization method was proposed in order to increase the density of the reactive hydroxyl groups on the surface of ceramic membranes, which were then amino-activated and treated with a crosslinker. The performance and the stability of the immobilized lipase were investigated as a function of the amount of the immobilized biocatalytst. Results showed that it is possible to immobilize lipase on a ceramic membrane without altering its catalytic performance (initial residual specific activity 93%), which remains constant after 6 reaction cycles.

Development of a Novel Immobilization Method by Using Microgels to Keep Enzyme in Hydrated Microenvironment in Porous Hydrophobic Membranes

Using colloidal polyacrylamide (PAAm) microgels as carriers, a novel strategy for covalent immobilization of enzymes maintained in hydrated microenvironment on/in a macroporous surface-functionalized hydrophobic polyvinylidene fluoride (PVDF) membrane is developed. The PAAm microgels are synthesized by inverse miniemulsion polymerization, and first the parameters are investigated which are suited to obtain particles in the desired size range, 100-200 nm, with narrow size distribution. Amino functions are then imparted to the microgels applying the Hofmann reaction. The modification is confirmed by Fourier-transform infrared spectroscopy analysis, ninhydrin test, and elemental analysis. In addition, functionalized microgels are characterized by dynamic light scattering. The amino-functionalized PAAm microgels are then immobilized on pre-modified PVDF membrane having aldehyde functionalities on the surface. Afterward, unreacted aldehyde groups still present on the membrane where quenched by ethanolamine and the enzyme lipase from Candida rugosa (LCR) is subsequently immobilized on the microgels loaded PVDF membrane via glutaraldehyde cross-linking, exploiting the free amino groups on immobilized microgels. Catalytic efficiency of LCR immobilized by this strategy is evaluated using para-nitrophenyl palmitate as substrate and compared with LCR directly immobilized on PVDF membrane without microgels. Results show that LCR immobilized by means of microgels exhibits better performance with a 2.3-fold higher specific biocatalytic activity.

Development of biohybrid immuno-selective membranes for target antigen recognition

Membranes are gaining increasing interest in solid-phase analytical assay and biosensors applications, in particular as functional surface for bioreceptors immobilization and stabilization as well as for the concentration of target molecules in microsystems. In this work, regenerated cellulose immuno-affinity membranes were developed and they were used for the selective capture of interleukin-6 (IL-6) as targeted antigen. Protein G was covalently linked on the membrane surface and it was successfully used for the oriented site-specific antibody immobilization. The antibody binding capacity of the protein G-coupled membrane was evaluated. The specific anti IL-6 antibody was immobilized and a quantitative analysis of the amount of IL-6 captured by the immuno-affinity membrane was performed. The immobilization procedure was optimized to eliminate the non-specific binding of antigen on the membrane surface. Additionally, the interaction between anti IL-6 antibody and protein G was stabilized by chemical cross-linking with glutaraldehyde and the capture ability of immuno-affinity membranes, with and without the cross-linker, was compared. The maximum binding capacity of the protein G-coupled membrane was 43.8µg/cm2 and the binding efficiency was 88%. The immuno-affinity membranes showed a high IL-6 capture efficiency at very low antigen concentration, up to a maximum of 91%, the amount of captured IL-6 increased linearly as increasing the initial concentration. The cross-linked surface retained the antigen binding capacity demonstrating its robustness in being reused, without antibody leakage or reduction in antibody binding capacity. The overall results demonstrated the possibility of a reliable application of the immuno-affinity membrane developed for biosensors and bioassays also in multiple use.

Biological Membranes and Biomimetic Artificial Membranes

This chapter aims at analyzing the biological membrane structure and composition with related functions and properties as a model for the development of bioartificial membrane systems as well as artificial membranes able to mimic the specificity and selectivity of the biomembrane. It therefore starts with some highlights on the biomembrane properties, and how these are achieved and performed. Then some example of natural systems will be identified as models for in vitro studies. Finally, artificial membrane systems able to mimic in vivo properties are described and highlights on future perspectives are outlined.

Integration of organic electrochemical transistors and immuno-affinity membranes for label-free detection of interleukin-6 in the physiological concentration range through antibody–antigen recognition

We demonstrate the label-free and selective detection of interleukin-6 (IL-6), a key cell-signaling molecule in biology and medicine, by integrating an OECT with an immuno-affinity regenerated cellulose membrane. The objective of the membrane is to increase the local concentration of IL-6 at the sensing electrode and, thereby, enhance the device response for concentrations falling within the physiological concentration range of cytokines. The OECT gate electrode is functionalized with an oligo(ethylene glycol)-terminated self-assembled alkanethiolate monolayer (SAM) for both the immobilization of anti IL-6 antibodies and the inhibition of non-specific biomolecule binding. The OECT gate/electrolyte interface is exploited for the selective detection of IL-6 through the monitoring of antigen-antibody binding events occurring at the gate electrode.

Phosphotriesterase-Magnetic Nanoparticle Bioconjugates with Improved Enzyme Activity in a Biocatalytic Membrane Reactor

The need to find alternative bioremediation solutions for organophosphate degradation pushed the research to develop technologies based on organophosphate degrading enzymes, such as phosphotriesterase. The use of free phosphotriesterase poses limits in terms of enzyme reuse, stability, and process development. The heterogenization of enzyme on a support and their use in bioreactors implemented by membranes seems a suitable strategy, thanks to the ability of membranes to compartmentalize, to govern mass transfer, and to provide a microenvironment with tuned physicochemical and structural properties. Usually, hydrophilic membranes are used since they easily guarantee the presence of water molecules needed for the enzyme catalytic activity. However, hydrophobic materials exhibit a larger shelf life and are preferred for the construction of filters and masks. Therefore, in this work, hydrophobic polyvinylidene fluoride (PVDF) porous membranes were used to develop biocatalytic membrane reactors (BMR). The phosphotriesterase-like lactonase (PLL) enzyme ( SsoPox triple mutant from S. solfataricus) endowed with thermostable phosphotriesterase activity was used as model biocatalyst. The enzyme was covalently bound directly to the PVDF hydrophobic membrane or it was bound to magnetic nanoparticles and then positioned on the hydrophobic membrane surface by means of an external magnetic field. Investigation of kinetic properties of the two BMRs and the influence of immobilized enzyme amount revealed that the performance of the BMR was mostly dependent on the amount of enzyme and its distribution on the immobilization support. Magnetic nanocomposite mediated immobilization showed a much better performance, with an observed specific activity higher than 90% compared to grafting of the enzyme on the membrane. Even though the present work focused on phosphotriesterase, it can be easily translated to other classes of enzymes and related applications.

1.1 From Biological Membranes to Artificial Biomimetic Membranes and Systems

This chapter aims at describing the biological membrane structure and composition with related functions and properties as a model for development of biohybrid membrane systems as well as synthetic membranes able to mimic the specificity and selectivity of the biomembrane. Besides, intriguing properties such as self-cleaning and self-healing will be discussed.