Biomass fractionation

The objective of this master's thesis was to develop a process to increase the value of residual fungal biomass as an animal feed. The increase in value is achieved by enriching the protein content in the biomass and potentially isolating other valuable fractions for productisation. In the literature part of this thesis the composition of fungal biomass and fungal cell wall and the factors affecting them during cultivation are presented. The possible processing options are also presented and evaluated. The soy protein and single cell protein product manufacturing processes are used as examples due to the lack of fungal biomass fractionation processes found in published literature. The second part of this thesis was performed by making laboratory experiments on the developed process, which consisted of acid hydrolysis with subsequent ethanol extraction. Chitin was precipitated from the acid hydrolysate filtrate. The experiments were conducted with three different hydrolysis temperatures and three different acid concentrations. The optimal hydrolysis conditions were 60 °C with 10 %-vol acid concentration. Optimal conditions in hydrolysis resulted in 30 % increase in protein content in the final biomass. The conceptual process was modelled to scale of 10 000 t/a biomass feed. The mass and energy balances were based on the laboratory experiments. Economic calculations were performed to determine the maximal capital expense while achieving 10 % internal rate of return for the investment. For the basic case the capital expense threshold was 25.8 M€. Four optional cases and parameter sensitivity analysis were performed to determine the effects of changes in the process. The chitin sales had the greatest impact of the individual parameters.

Tasasähköverkon modulaarisen vaihtosuuntaajan rakenne- ja kustannusanalyysi

Diplomityössä tarkastellaan tapausta, jossa tasasähköverkon vaihtosuuntaaja toteutetaan kolmella yksivaiheisella suuntaajalla yhden kolmivaiheisen sijaan. Modulaarinen rakenne mahdollistaa sen, että osa vaiheista voitaisiin sammuttaa kun kuormitus on erittäin pientä eikä kuorma ole kolmivaiheista. Galvaaninen erotus oletetaan tehtävän erottavalla DC-DC -konvertterilla, joka mahdollistaa samalla vaihtosuuntaajan DC-syöttöjännitteen muuttamisen. Työssä tutkitaan, mitkä tekijät vaikuttavat DC-syöttöjännitteen valintaan sekä tehdään valinta kyseisen jännitteen arvolle. Valittua jännitetasoa käytetään vaihtosuuntaajan suunnittelemiseen. Vaihtosuuntaajan rakennetta tarkastellaan vaihtosuuntaussillan kytkinkomponenttien sekä lähtöjännitteen suotimen osalta. Yksivaiheiselle moduulille määritetään optimaalinen teholuokka investointi- ja käyttökustannusten kannalta. Tarkastelu pitää sisällään erilaisten transistoritekniikkojen, kytkentätaajuuksien sekä kahden suodintyypin vaikutuksen kustannustehokkuuteen.

Studies on chemoenzymatic synthesis: Lipase-catalyzed acylation in multistep organic synthesis

In this thesis, biocatalysis is defined as the science of using enzymes as catalysts in organic synthesis. Environmental aspects and the continuously expanding repertoire of available enzymes have firmly established biocatalysis as a prominent means of chemo-, regio- and stereoselective synthesis. Yet, no single methodology can solve all the challenges faced by a synthetic chemist. Therefore, the knowledge and the skills to combine different synthetic methods are relevant. Lipases are highly useful enzymes in organic synthesis. In this thesis, an effort is being made to form a coherent picture of when and how can lipases be incorporated into nonenzymatic synthesis. This is attempted both in the literature review and in the discussion of the results presented in the original publications contained in the thesis. In addition to lipases, oxynitrilases were also used in the work. The experimental part of the thesis comprises of the results reported in four peer-reviewed publications and one manuscript. Selected amines, amino acids and sugar-derived cyanohydrins or their acylated derivatives were each prepared in enantio- or diastereomerically enriched form. Where applicable, attempts were made to combine the enzymatic reactions to other synthetic steps either by the application of completely separate sequential reactions with isolated intermediates (kinetic and functional kinetic resolution of amines), simultaneously occurring reactions without intermediate isolation (dynamic kinetic resolution of amino acid esters) or sequential reactions but without isolating the intermediates (hydrocyanation of sugar aldehydes with subsequent diastereoresolution). In all cases, lipase-catalyzed acylation was the key step by which stereoselectivity was achieved. Lipase from Burkholderia cepacia was a highly selective enzyme with each substrate category, but careful selection of the acyl donor and the solvent was important as well.

Matched case-control study evaluating the frequency of the main agents associated with neonatal diarrhea in piglets

A case-control study was carried out in litters of 1 to 7-day-old piglets to identify the main infectious agents involved with neonatal diarrhea in pigs. Fecal samples (n=276) from piglets were collected on pig farms in the State of Rio Grande do Sul, Brazil, from May to September 2007. Litters with diarrhea were considered cases (n=129) and normal litters (n=147) controls. The samples were examined by latex agglutination test, PAGE, conventional isolating techniques, ELISA, PCR, and microscopic methods in order to detect rotavirus, bacterial pathogens (Escherichia coli, Clostridium perfringens type A and C, and Clostridium difficile), and parasites (Coccidian and Cryptosporidium spp.). Outbreaks of diarrhea were not observed during sampling. At least one agent was detected in fecal samples on 25 out of 28 farms (89.3%) and in 16 farms (57.1%) more than one agent was found. The main agents diagnosed were Coccidia (42.86%) and rotavirus (39.29%). The main agents identified in litters with diarrhea were Clostridium difficile (10.6%), Clostridium perfringens type A (8.8%) and rotavirus (7.5%); in control litters, Clostridium difficile (16.6%) and Coccidian (8.5%). Beta hemolytic Escherichia coli and Clostridium perfringens type C were not detected. When compared with controls, no agent was significantly associated with diarrhea in case litters. These findings stress the need for caution in the interpretation of laboratorial diagnosis of mild diarrhea in neonatal pigs, as the sole detection of an agent does not necessarily indicate that it is the cause of the problem.

Serologic and molecular diagnostic and bioassay in mice for detection of Toxoplasma gondii in free ranges chickens from Pantanal of Mato Grosso do Sul

The aim of this study was to investigate the occurrence of Toxoplasma gondii and compare the results obtained in the Modified Agglutination Test (MAT), Polimerase Chain Reaction (PCR) and bioassay in mice. In order to accomplish this, 40 free-range chickens from eight farms in neighboring areas to the Pantanal in Nhecolândia, Mato Grosso do Sul, were euthanized and blood samples, brain and heart were collected. The occurrence of anti-T. gondii antibodies found in chickens was 67.5% (27 samples), considering as a cutoff point the dilution 1:5. Among the samples analyzed, 7 (25.9%) were positive in the dilution 1:5, 3 (11.1%) in 1:10, 2 (7.4%) in 1:20, 3 (11.1%) in 1:320, 1 ( 3.7%) in 1:640, 3 (11.1%) in 1:1280, 2 (7.4%) in 1:2560, 4 (14.8%) in 1:5120 and 2 (7.4%) in 1:10.240. From the mixture of tissue samples (brain and heart) from the chickens analyzed, 16 (40%) presented electrophoretic bands compatible with T. gondii by PCR (gene B1). In the comparison of techniques, 59.26% positivity in PCR was revealed among animals that were seropositive in MAT (cutoff 1:5). From 141 inoculated mice, six (4.44%) died of acute toxoplasmosis between 15 and 23 days after inoculation. Surviving mice were sacrificed at 74 days after inoculation, and a total of 28 cysts were found in the brains of 10 distinct groups. From the seropositive hens, 27 bioassays were performed and 11 (40.7%) isolates were obtained. A greater number of isolations happened in mice that were inoculated with tissues from chickens that had high titers for anti-T. gondii antibodies. Chronic infection in mice was observed in nine groups (33.3%) from five different properties. Among the surviving mice, 25.6% were positive for T. gondii in MAT (1:25). From mice positive in PCR, 87.5% were also positive in MAT. Among the PCR-negative mice, 5.2% were positive for T. gondii in MAT. It can be concluded through this study that the occurrence of infecton by T. gondii in the rural properties studied was high, that PCR directed to gene B1 does not confirm the viability of the parasite, but it can be used as a screening method for the selection of chickens infected by T. gondii, that the animals with titer greater than 10 must be prioritized for the selection of animals for bioassay, since for them, the chances of isolating the parasite are greater and that seroconversion in experimentally infected mice is not a good indicator for isolating the agent.

Recovery of biomass-derived valuable compounds using chromatographic and membrane separations

Utilization of biomass-based raw materials for the production of chemicals and materials is gaining an increasing interest. Due to the complex nature of biomass, a major challenge in its refining is the development of efficient fractionation and purification processes. Preparative chromatography and membrane filtration are selective, energy-efficient separation techniques which offer a great potential for biorefinery applications. Both of these techniques have been widely studied. On the other hand, only few process concepts that combine the two methods have been presented in the literature. The aim of this thesis was to find the possible synergetic effects provided by combining chromatographic and membrane separations, with a particular interest in biorefinery separation processes. Such knowledge could be used in the development of new, more efficient separation processes for isolating valuable compounds from complex feed solutions that are typical for the biorefinery environment. Separation techniques can be combined in various ways, from simple sequential coupling arrangements to fully-integrated hybrid processes. In this work, different types of combined separation processes as well as conventional chromatographic separation processes were studied for separating small molecules such as sugars and acids from biomass hydrolysates and spent pulping liquors. The combination of chromatographic and membrane separation was found capable of recovering high-purity products from complex solutions. For example, hydroxy acids of black liquor were successfully recovered using a novel multistep process based on ultrafiltration and size-exclusion chromatography. Unlike any other separation process earlier suggested for this challenging separation task, the new process concept does not require acidification pretreatment, and thus it could be more readily integrated into a pulp-mill biorefinery. In addition to the combined separation processes, steady-state recycling chromatography, which has earlier been studied for small-scale separations of high-value compounds only, was found a promising process alternative for biorefinery applications. In comparison to conventional batch chromatography, recycling chromatography provided higher product purity, increased the production rate and reduced the chemical consumption in the separation of monosaccharides from biomass hydrolysates. In addition, a significant further improvement in the process performance was obtained when a membrane filtration unit was integrated with recycling chromatography. In the light of the results of this work, separation processes based on combining membrane and chromatographic separations could be effectively applied for different biorefinery applications. The main challenge remains in the development of inexpensive separation materials which are resistant towards harsh process conditions and fouling.

Using the best linear predictor (BLP) in the selection between and among half-sib progenies of the CMS-39 maize population

Data of corn ear production (kg/ha) of 196 half-sib progenies (HSP) of the maize population CMS-39 obtained from experiments carried out in four environments were used to adapt and assess the BLP method (best linear predictor) in comparison with to the selection among and within half-sib progenies (SAWHSP). The 196 HSP of the CMS-39 population developed by the National Center for Maize and Sorghum Research (CNPMS-EMBRAPA) were related through their pedigree with the recombined progenies of the previous selection cycle. The two methodologies used for the selection of the twenty best half-sib progenies, BLP and SAWHSP, led to similar expected genetic gains. There was a tendency in the BLP methodology to select a greater number of related progenies because of the previous generation (pedigree) than the other method. This implies that greater care with the effective size of the population must be taken with this method. The SAWHSP methodology was efficient in isolating the additive genetic variance component from the phenotypic component. The pedigree system, although unnecessary for the routine use of the SAWHSP methodology, allowed the prediction of an increase in the inbreeding of the population in the long term SAWHSP selection when recombination is simultaneous to creation of new progenies.

Insect juvenile hormone: from "status quo" to high society

Juvenile hormone (JH) exerts pleiotropic functions during insect life cycles. The regulation of JH biosynthesis by neuropeptides and biogenic amines, as well as the transport of JH by specific binding proteins is now well understood. In contrast, comprehending its mode of action on target organs is still hampered by the difficulties in isolating specific receptors. In concert with ecdysteroids, JH orchestrates molting and metamorphosis, and its modulatory function in molting processes has gained it the attribute "status quo" hormone. Whereas the metamorphic role of JH appears to have been widely conserved, its role in reproduction has been subject to many modifications. In many species, JH stimulates vitellogenin synthesis and uptake. In mosquitoes, however, this function has been transferred to ecdysteroids, and JH primes the ecdysteroid response of developing follicles. As reproduction includes a variety of specific behaviors, including migration and diapause, JH has come to function as a master regulator in insect reproduction. The peak of pleiotropy was definitely reached in insects exhibiting facultative polymorphisms. In wing-dimorphic crickets, differential activation of JH esterase determines wing length. The evolution of sociality in Isoptera and Hymenoptera has also extensively relied on JH. In primitively social wasps and bumble bees, JH integrates dominance position with reproductive status. In highly social insects, such as the honey bee, JH has lost its gonadotropic role and now regulates division of labor in the worker caste. Its metamorphic role has been extensively explored in the morphological differentiation of queens and workers, and in the generation of worker polymorphism, such as observed in ants.

Minimizing Circulating Current in Parallel-Connected Photovoltaic Inverters

Parallel-connected photovoltaic inverters are required in large solar plants where it is not economically or technically reasonable to use a single inverter. Currently, parallel inverters require individual isolating transformers to cut the path for the circulating current. In this doctoral dissertation, the problem is approached by attempting to minimize the generated circulating current. The circulating current is a function of the generated common-mode voltages of the parallel inverters and can be minimized by synchronizing the inverters. The synchronization has previously been achieved by a communication link. However, in photovoltaic systems the inverters may be located far apart from each other. Thus, a control free of communication is desired. It is shown in this doctoral dissertation that the circulating current can also be obtained by a common-mode voltage measurement. A control method based on a short-time switching frequency transition is developed and tested with an actual photovoltaic environment of two parallel inverters connected to two 5 kW solar arrays. Controls based on the measurement of the circulating current and the common-mode voltage are generated and tested. A communication-free method of controlling the circulating current between parallelconnected inverters is developed and verified.

Characterisation of Cladonia rangiferina transcriptome and genome, and the effects of dehydration and rehydration on its gene expression

Lichens are symbiotic organisms, which consist of the fungal partner and the photosynthetic partner, which can be either an alga or a cyanobacterium. In some lichen species the symbiosis is tripartite, where the relationship includes both an alga and a cyanobacterium alongside the primary symbiont, fungus. The lichen symbiosis is an evolutionarily old adaptation to life on land and many extant fungal species have evolved from lichenised ancestors. Lichens inhabit a wide range of habitats and are capable of living in harsh environments and on nutrient poor substrates, such as bare rocks, often enduring frequent cycles of drying and wetting. Most lichen species are desiccation tolerant, and they can survive long periods of dehydration, but can rapidly resume photosynthesis upon rehydration. The molecular mechanisms behind lichen desiccation tolerance are still largely uncharacterised and little information is available for any lichen species at the genomic or transcriptomic level. The emergence of the high-throughput next generation sequencing (NGS) technologies and the subsequent decrease in the cost of sequencing new genomes and transcriptomes has enabled non-model organism research on the whole genome level. In this doctoral work the transcriptome and genome of the grey reindeer lichen, Cladonia rangiferina, were sequenced, de novo assembled and characterised using NGS and traditional expressed sequence tag (EST) technologies. RNA extraction methods were optimised to improve the yield and quality of RNA extracted from lichen tissue. The effects of rehydration and desiccation on C. rangiferina gene expression on whole transcriptome level were studied and the most differentially expressed genes were identified. The secondary metabolites present in C. rangiferina decreased the quality – integrity, optical characteristics and utility for sensitive molecular biological applications – of the extracted RNA requiring an optimised RNA extraction method for isolating sufficient quantities of high-quality RNA from lichen tissue in a time- and cost-efficient manner. The de novo assembly of the transcriptome of C. rangiferina was used to produce a set of contiguous unigene sequences that were used to investigate the biological functions and pathways active in a hydrated lichen thallus. The de novo assembly of the genome yielded an assembly containing mostly genes derived from the fungal partner. The assembly was of sufficient quality, in size similar to other lichen-forming fungal genomes and included most of the core eukaryotic genes. Differences in gene expression were detected in all studied stages of desiccation and rehydration, but the largest changes occurred during the early stages of rehydration. The most differentially expressed genes did not have any annotations, making them potentially lichen-specific genes, but several genes known to participate in environmental stress tolerance in other organisms were also identified as differentially expressed.

Development of packaging cell lines for rescuing BAV2 viral vectors

The construction of adenovirus vectors for cloning and foreign gene expression requires packaging cell lines that can complement missing viral functions caused by sequence deletions and/or replacement with foreign DNA sequences. In this study, packaging cell lines were designed to provide in trans the missing bovine adenovirus functions, so that recombinant viruses could be generated. Fetal bovine kidney and lUng cells, acquired at the trimester term from a pregnant cow, were tranfected with both digested wild type BAV2 genomic DNA and pCMV-EI. The plasmid pCMV-EI was specifically constructed to express El of BAV2 under the control of the cytomegalovirus enhancer/promoter (CMV). Selection for "true" transformants by continuous passaging showed no success in isolating immortalised cells, since the cells underwent crisis resulting in complete cell death. Moreover, selection for G418 resistance, using the same cells, also did not result in the isolation of an immortalised cell line and the same culture-collapse event was observed. The lack of success in establishing an immortalised cell line from fetal tissue prompted us to transfect a pre-established cell line. We began by transfecting MDBK (Mardin-Dardy bovine kidney) cells with pCMV-El-neo, which contain the bacterial selectable marker neo gene. A series of MDBK-derived cell lines, that constitutively express bovine adenoviral (BAV) early region 1 (El), were then isolated. Cells selected for resistance to the drug G418 were isolated collectively for full characterisation to assess their suitability as packaging cell lines. Individual colonies were isolated by limiting dilution and further tested for El expression and efficiency of DNA uptake. Two cell lines, L-23 and L-24, out of 48 generated foci tested positive for £1 expression using Northern Blot analysis. DNA uptake studies, using both lipofectamine and calcium phosphate methods, were performed to compare these cells, their parental MDBK cells, 8 and the unrelated human 293 cells as a benchmark. The results revealed that the new MDBKderived clones were no more efficient than MDBK cells in the transient expression of transfected DNA and that they were inferior to 293 cells, when using lacZ as the reporter gene. In view of the inherently poor transfection efficiency of MDBK cells and their derivatives, a number of other bovine cells were investigated for their potential as packaging cells. The cell line CCL40 was chosen for its high efficiency in DNA uptake and subsequently transfected with the plasmid vector pCMV El-neo. By selection with the drug G418, two cell lines were isolated, ProCell 1 and ProCell 2. These cell lines were tested for El expression, permissivity to BAV2 and DNA uptake efficiency, revealing a DNA uptake efficiency of 37 % , comparable to that of CCL40. Attempts to rescue BAV2 mutants carrying the lacZ gene in place of £1 or £3 were carried out by co-transfecting wild type viral DNA with either the plasmid pdlElE-Z (which contains BAV2 sequences from 0% to 40.4% with the lacZ gene in place of the £1 region from 1.1% to 8.25%) or with the plasmid pdlE3-5-Z (which contains BAV2 sequences from 64.8% to 100% with the lacZ gene in place of the E3 region from 75.8% to 81.4%). These cotransfections did not result in the generation of a viral mutant. The lack of mutant generation was thought to be caused by the relative inefficiency ofDNA uptake. Consequently, cosBAV2, a cosmid vector carrying the BAV2 genome, was modified to carry the neo reporter gene in place of the £3 region from 75.8% to 81.4%. The use of a single cosmid vector earring the whole genome would eliminate the need for homologous recombination in order to generate a viral vector. Unfortunately, the transfection of cosBAV2- neo also did not result in the generation of a viral mutant. This may have been caused by the size of the £3 deletion, where excess sequences that are essential to the virus' survival might have been deleted. As an extension to this study, the spontaneous E3 deletion, accidently discovered in our viral stock, could be used as site of foreign gene insertion.

Autonomic and Electrophysiological Correlates of Cognitive Control in Aging

This thesis tested a model of neurovisceral integration (Thayer & Lane, 2001) wherein parasympathetic autonomic regulation is considered to play a central role in cognitive control. We asked whether respiratory sinus arrhythmia (RSA), a parasympathetic index, and cardiac workload (rate pressure product, RPP) would influence cognition and whether this would change with age. Cognitive control was measured behaviourally and electrophysiologically through the error-related negativity (ERN) and error positivity (Pe). The ERN and Pe are thought to be generated by the anterior cingulate cortex (ACC), a region involved in regulating cognitive and autonomic control and susceptible to age-related change. In Study 1, older and younger adults completed a working memory Go/NoGo task. Although RSA did not relate to performance, higher pre-task RPP was associated with poorer NoGo performance among older adults. Relations between ERN/Pe and accuracy were indirect and more evident in younger adults. Thus, Study 1 supported the link between cognition and autonomic activity, specifically, cardiac workload in older adults. In Study 2, we included younger adults and manipulated a Stroop task to clarify conditions under which associations between RSA and performance will likely emerge. We varied task parameters to allow for proactive versus reactive strategies, and motivation was increased via financial incentive. Pre-task RSA predicted accuracy when response contingencies required maintenance of a specific item in memory. Thus, RSA was most relevant when performance required proactive control, a metabolically costly strategy that would presumably be more reliant on autonomic flexibility. In Study 3, we included older adults and examined RSA and proactive control in an additive factors framework. We maintained the incentive and measured fitness. Higher pre-task RSA among older adults was associated with greater accuracy when proactive control was needed most. Conversely, performance of young women was consistently associated with fitness. Relations between ERN/Pe and accuracy were modest; however, isolating ACC activity via independent component analysis allowed for more associations with accuracy to emerge in younger adults. Thus, performance in both groups appeared to be differentially dependent on RSA and ACC activation. Altogether, these data are consistent with a neurovisceral integration model in the context of cognitive control.

L'autonomie de la clause compromissoire en matière d'arbitrage international

"Mémoire présenté à la Faculté des études supérieures en vue de l'obtention du grade de maîtrise en droit (LL.M.) (Option: droit des affaires )"

Biocompatibilité des microcapsules d'alginate : purification d'alginate, réaction immunitaire de l'hôte et protection du receveur

L’immuno-isolation des îlots de Langerhans est proposée comme moyen d’effectuer des transplantations sans prise d’immunosuppresseurs par le patient. Cette immuno-isolation, par l’entremise d’une microcapsule composée d’alginate et de poly-L-lysine (microcapsule APA), protège le greffon d’une éventuelle attaque du système immunitaire du receveur grâce à sa membrane semi-perméable. Cette membrane empêche le système immunitaire du receveur de pénétrer la microcapsule tout en laissant diffuser librement les nutriments, le glucose et l’insuline. Avant l’application de cette technique chez l’humain, quelques défis doivent encore être relevés, dont la biocompatibilité de ce système. La biocompatibilité fait ici référence à la biocompatibilité du biomatériau utilisé pour la fabrication des microcapsules, l’alginate, mais aussi la biocompatibilité des microcapsules reliée à leur stabilité. En effet, il a été remarqué que, lors d’implantation in vivo de microcapsules fabriquées avec de l’alginate non purifiée, ceci induisait un phénomène nommé Réaction de l’Hôte contre la Microcapsule (RHM). De plus, il est connu que la stabilité des microcapsules APA peut influencer leur biocompatibilité puisqu’une microcapsule endommagée ou brisée pourrait laisser s’échapper les cellules du greffon chez le receveur. Nous croyons qu’une compréhension des processus d’initiation de la RHM en fonction de l’efficacité des procédés de purification d’alginate (et donc des quantités de contaminants présents dans l’alginate) ainsi que l’augmentation de la stabilité des microcapsules APA pourront améliorer la biocompatibilité de ce dispositif, ce que tente de démontrer les résultats présentés dans cette thèse. En effet, les résultats obtenus suggèrent que les protéines qui contaminent l’alginate jouent un rôle clé dans l’initiation de la RHM et qu’en diminuant ces quantités de protéines par l’amélioration des procédés de purification d’alginate, on améliore la biocompatibilité de l’alginate. Afin d’augmenter la stabilité des microcapsules APA, nous décrivons une nouvelle technique de fabrication des microcapsules qui implique la présence de liaisons covalentes. Ces nouvelles microcapsules APA réticulées sont très résistantes, n’affectent pas de façon négative la survie des cellules encapsulées et confinent les cellules du greffon à l’intérieur des microcapsules. Cette dernière caractéristique nous permet donc d’augmenter la biocompatibilité des microcapsules APA en protégeant le receveur contre les cellules du greffon.

Développement de papier bioactif par couchage à grande échelle d’enzymes immobilisées par microencapsulation

L’objectif principal de cette recherche est de contribuer au développement de biocapteurs commerciaux utilisant des surfaces de papier comme matrices d’immobilisation, capables de produire un signal colorimétrique perceptible dans les limites sensorielles humaines. Ce type de biocapteur, appelé papier bioactif, pourrait servir par exemple à la détection de substances toxiques ou d’organismes pathogènes. Pour atteindre l’objectif énoncé, ce travail propose l’utilisation de systèmes enzymatiques microencapsulés couchés sur papier. Les enzymes sont des catalyseurs biologiques dotés d’une haute sélectivité, et capables d'accélérer la vitesse de certaines réactions chimiques spécifiques jusqu’à des millions des fois. Les enzymes sont toutefois des substances très sensibles qui perdent facilement leur fonctionnalité, raison pour laquelle il faut les protéger des conditions qui peuvent les endommager. La microencapsulation est une technique qui permet de protéger les enzymes sans les isoler totalement de leur environnement. Elle consiste à emprisonner les enzymes dans une sphère poreuse de taille micrométrique, faite de polymère, qui empêche l’enzyme de s’echapper, mais qui permet la diffusion de substrats à l'intérieur. La microencapsulation utilisée est réalisée à partir d’une émulsion contenant un polymère dissous dans une phase aqueuse avec l’enzyme désirée. Un agent réticulant est ensuite ajouté pour provoquer la formation d'un réseau polymérique à la paroi des gouttelettes d'eau dans l'émulsion. Le polymère ainsi réticulé se solidifie en enfermant l’enzyme à l'intérieur de la capsule. Par la suite, les capsules enzymatiques sont utilisées pour donner au papier les propriétés de biocapteur. Afin d'immobiliser les capsules et l'enzyme sur le papier, une méthode courante dans l’industrie du papier connu sous le nom de couchage à lame est utilisée. Pour ce faire, les microcapsules sont mélangées avec une sauce de couchage qui sera appliquée sur des feuilles de papier. Les paramètres de viscosité i de la sauce et ceux du couchage ont été optimisés afin d'obtenir un couchage uniforme répondant aux normes de l'industrie. Les papiers bioactifs obtenus seront d'abord étudiés pour évaluer si les enzymes sont toujours actives après les traitements appliqués; en effet, tel que mentionné ci-dessus, les enzymes sont des substances très sensibles. Une enzyme très étudiée et qui permet une évaluation facile de son activité, connue sous le nom de laccase, a été utilisée. L'activité enzymatique de la laccase a été évaluée à l’aide des techniques analytiques existantes ou en proposant de nouvelles techniques d’analyse développées dans le laboratoire du groupe Rochefort. Les résultats obtenus démontrent la possibilité d’inclure des systèmes enzymatiques microencapsulés sur papier par couchage à lame, et ce, en utilisant des paramètres à grande échelle, c’est à dire des surfaces de papier de 0.75 x 3 m2 modifiées à des vitesses qui vont jusqu’à 800 m/min. Les biocapteurs ont retenu leur activité malgré un séchage par évaporation de l’eau à l’aide d’une lampe IR de 36 kW. La microencapsulation s’avère une technique efficace pour accroître la stabilité d’entreposage du biocapteur et sa résistance à l’exposition au NaN3, qui est un inhibiteur connu de ce biocapteur. Ce projet de recherche fait partie d'un effort national visant à développer et à mettre sur le marché des papiers bioactifs; il est soutenu par Sentinel, un réseau de recherche du CRSNG.