Dominic Rochefort

Electron transfer mediator systems for bleaching of paper pulp

The participation of biological agents in pulp bleaching systems has received a lot of attention from research teams around the world, driven by the environmental benefits that biobleaching could bring. Nature showed us the ability of some of its agents, such as wood-decaying fungi, to delignify and bleach wood and wood pulp. What we need to do is to enhance the efficiency of such agents to make them cope with the fast pace of our modern pulp mills. To do so, a profound understanding of the biobleaching system is required. Our efforts to discover new efficient mediators for the laccase-mediator system (LMS) brought us to use several techniques to analyse the reactions involved in mediated enzymatic delignification. Mostly based on electrochemistry, these techniques are reviewed in this paper, along with key results. Cyclic voltammetry was used to characterize electron transfer rates between each element of the LMS. We found, along with other authors, that the mediator redox potential has a great influence on its efficiency. We used bulk electrolysis to simulate the oxidative action of laccase on mediators and model compounds of lignin. Such electrolysis techniques allowed us to study mediated lignin oxidation outside of normal laccase working conditions. Finally, an electrolysis-based method for mediated pulp delignification that we developed, based upon our research on biobleaching, is presented.

Printing of polymer microcapsules for enzyme immobilization on paper substrate.

Poly(ethyleneimine) (PEI) microcapsules containing laccase from Trametes hirsuta (ThL) and Trametes versicolor (TvL) were printed onto paper substrate by three different methods: screen printing, rod coating, and flexo printing. Microcapsules were fabricated via interfacial polycondensation of PEI with the cross-linker sebacoyl chloride, incorporated into an ink, and printed or coated on the paper substrate. The same ink components were used for three printing methods, and it was found that laccase microcapsules were compatible with the ink. Enzymatic activity of microencapsulated TvL was maintained constant in polymer-based ink for at least eight weeks. Thick layers with high enzymatic activity were obtained when laccase-containing microcapsules were screen printed on paper substrate. Flexo printed bioactive paper showed very low activity, since by using this printing method the paper surface was not fully covered by enzyme microcapsules. Finally, screen printing provided a bioactive paper with high water-resistance and the highest enzyme lifetime.

Development of prototypes of bioactive packaging materials based on immobilized bacteriophages for control of growth of bacterial pathogens in foods.

Due to lack of adequate control methods to prevent contamination in fresh produce and growing consumer demand for natural products, the use of bacteriophages has emerged as a promising approach to enhance safety of these foods. This study sought to control Listeria monocytogenes in cantaloupes and RTE meat and Escherichia coli O104:H4 in alfalfa seeds and sprouts under different storage conditions by using specific lytic bacteriophage cocktails applied either free or immobilized. Bacteriophage cocktails were introduced into prototypes of packaging materials using different techniques: i) immobilizing on positively charged modified cellulose membranes, ii) impregnating paper with bacteriophage suspension, and iii) encapsulating in alginate beads followed by application of beads onto the paper. Phage-treated and non-treated samples were stored for various times and at temperatures of 4°C, 12°C or 25°C. In cantaloupe, when free phage cocktail was added, L. monocytogenes counts dropped below the detection limit of the plating technique (<1 log CFU/g) after 5 days of storage at both 4°C and 12°C. However, at 25°C, counts below the detection limit were observed after 3 and 6h and a 2-log CFU/g reduction in cell numbers was seen after 24h. For the immobilized Listeria phage cocktail, around 1-log CFU/g reduction in the Listeria count was observed by the end of the storage period for all tested storage temperatures. For the alfalfa seeds and sprouts, regardless of the type of phage application technique (spraying of free phage suspension, bringing in contact with bacteriophage-based materials (paper coated with encapsulated bacteriophage or impregnated with bacteriophage suspension)), the count of E. coli O104:H4 was below the detection limit (<1 log CFU/g) after 1h in seeds and about a 1-log cycle reduction in E. coli count was observed on the germinated sprouts by day 5. In ready-to-eat (RTE) meat, LISTEX™ P100, a commercial phage product, was able to significantly reduce the growth of L. monocytogenes at both storage temperatures, 4°C and 10°C, for 25 days regardless of bacteriophage application format (immobilized or non-immobilized (free)). In conclusion, the developed phage-based materials demonstrated significant antimicrobial effect, when applied to the artificially contaminated foods, and can be used as prototypes for developing bioactive antimicrobial packaging materials capable of enhancing the safety of fresh produce and RTE meat.

Comparison of emulsion and vibration nozzle methods for microencapsulation of laccase and glucose oxidase by interfacial reticulation of poly(ethyleneimine)

Microcapsules for enzyme immobilization were successfully fabricated via interfacial cross-linking of poly(ethyleneimine) (PEI). A method based on laminar jet break-up technique using a commercial instrument developed to produce alginate beads is reported for the first time for production of PEI microcapsules. The diameter, wall thickness and pore size of membranes were obtained from confocal laser scanning microscopy by labelling PEI and proteins. The composition of membranes was analysed by elemental analysis. Larger microcapsules (ca 200 µm diameter) were obtained with the encapsulation device. In comparison, the emulsion method produced smaller capsules (ca 20 µm diameter) but with a wider size distribution. Encapsulation efficiency for both methods was analysed by bicinchoninic acid and fluorescence assays, yielding efficiencies of 94 ± 2% and 83 ± 3% for the emulsion method and encapsulation device, respectively. Glucose oxidase from Aspergillus Niger and Laccase from Trametes Versicolor were encapsulated by both microencapsulation methods and their activities were compared.

Characterisation and applications of microcapsules obtained by interfacial polycondensation

This review highlights the materials, mechanisms and applications of microencapsulation by interfacial polycondensation in different areas. This technology entraps active ingredients inside microcapsules/microspheres, having an average diameter ranging from nanosize to several 100 µ. Polycondensation reactions take place at the boundary of two phases to form the shells of microcapsules or matrix microspheres. The emulsion can be classified into three types: water-in-oil, oil-in-water and oil-in-oil. According to the hydrophilic–lipophilic property of core phase, different active substances, such as proteins, enzymes, insecticides, herbicides, vitamins, catalysts, drugs, essential oils, dyes and phase change materials, have been successfully incorporated into different microcapsules/microspheres. Based on the shell-forming materials, this technology is capable of preparing polyamine, polyurea, polyurethane, polythiourea, polyester, polyepoxide, polyacrylamide and polysiloxane microcapsules. Over the past two decades, microcapsules prepared by interfacial polycondensation have been widely used in carbonless paper, cosmetics, pharmacy, agriculture, energy storage/transfer, thermal insulation/regulation and information and magnetic recording.

Development of an enzymatic microreactor based on microencapsulated laccase with off-line capillary electrophoresis for measurement of oxidation reactions

Microencapsulation is used here as a new technique to immobilize enzymes in a microreactor coupled off-line to capillary electrophoresis (CE), allowing the determination of enzymatic reaction products. The redox enzyme laccase was encapsulated using the method of interfacial cross-linking of poly(ethyleneimine) (PEI). The 50 microm diameter capsules were slurry packed from a suspension into a capillary-sized reactor made easily and quickly from a short length of 530 microm diameter fused-silica tubing. The volume of the bed of laccase microcapsules in the microreactor was in the order of 1.1 microL through which 50 microL of the substrate o-phenylenediamine (OPD) was flowed. The oxidation product 2,3-diaminophenazine (DAP) and the remaining OPD were quantified by CE in a pH 2.5 phosphate buffer. Peak migration time reproducibility was in the order of 0.4% RSD and peak area reproducibility was less than 1.7% RSD within the same day. Using the OPD peak area calibration curve, a conversion efficiency of 48% was achieved for a 2-min oxidation reaction in the microreactor.

Oxidation of lignin model compounds by organic and transition metal-based electron transfer mediators

We have studied the oxidation of lignin model compounds by organic and transition metal-based mediators using either an enzyme or an electrolysis cell as the mediator oxidizing agent. Electrolysis of inorganic mediator seems a promising technology for pulp delignification.

Mesomorphic and ion conducting properties of dialkyl(1,4-phenylene)diimidazolium salts

A new family of dialkyl(1,4-phenylene)diimidazolium salts has been synthesized. Salts containing triflate and bis(trifluoromethanesulfonyl)imide anions and different alkyl chains have been prepared. The molecular structures of didodecyl(1,4-phenylene)diimidazolium triflate and bis(trifluoromethylsulfonyl)imide salts were determined by single crystal X-ray diffraction, and the mesomorphic and ionic conducting properties of all these compounds were investigated and are reported here.

Electrochemical Oxidation of Transition Metal-Based Mediators for Pulp Delignification

In order to depolymerize and remove the lignin that remains in paper pulp following the pulping process, the pulp must be bleached. Environmental considerations and system closure requirements have created a need for replacement of the traditionally used chlorine bleaching technologies. Mediated laccase oxidation of residual lignin in pulp has gained in popularity during the last 10 years and could be considered as a good alternative bleaching method. The mechanisms leading to mediated lignin oxidation are still unclear. To clarify the role of the mediator during mediated kraft pulp delignification and to increase the efficiency of the system, we have studied the oxidation of K 4 Mo(CN) 8 (MoCn) mediator in an electrolysis cell prior to its reaction with pulp. The electrochemical oxidation of MoCN gave similar delignification levels to enzymatic oxidation under similar conditions, while the iron-based mediators gave better delignification than enzymatic oxidation. The electrodelignification setup allowed us to study the effect of pH and oxygen on the lignin degradation. We also report on the use and efficiency of high formal potential mediators that are suspected to be good lignin oxidants hut which are not readily oxidized by the laccase. By studying the charge transferred to the solution to achieve mediator oxidation we were able to show that the mediator acts as catalyst, i.e., is being continuously regenerated throughout the process, to perform efficient pulp delignification.

Electroactive imidazolium salts based on 1,4-dimethoxybenzene redox groups: synthesis and electrochemical characterisation

Three new redox-active ionic salts have been synthesised based on 1,4-dimethoxybenzene (1) and 2,5-di-tert-butyl-1,4-dimethoxybenzene (2 and 3). These imidazolium salts are the first organic redox-active groups to be incorporated into an ionic liquid structure. There are no negative effects on the transport properties by incorporating 2,5-di-tert-butyl-1,4-dimethoxybenzene into an electroactive imidazolium salt. The thermal stability and oxidation potential have been increased.