The objective of this study was to investigate the possibility of recycling enzymes during saccharification of cellulose for the production of ethanol from woodchips. To make enzyme recycling feasible and economical when woodchips are processed for ethanol, the lignin in the wood is to be removed before the enzymes are added. Since enzymes constitute a major part of the input costs, second only to the feedstock, the ability to reuse the enzymes could lead to a considerable decrease in the production cost of ethanol. Tulip poplar woodchips were selected as the feedstock. Different delignification methods with recovery of byproducts were investigated. Alkali extraction, using dilute NaOH for the removal of lignin after steam pretreatment, was used as the base case against which all other processes were compared. Recovery of furfural and methanol, produced during the pretreatment of the woodchips, for sale as byproducts was one modification to the alkali extraction process that was investigated. The conversion of xylose to furfural and the recovery of the furfural as a byproduct was the third case explored. Solvent extraction using a 50:50 ethanol-water mixture instead of extraction with NaOH was the fourth case examined. Process flow sheets were then developed to recycle the enzymes during the hydrolysis and fermentation of this prehyrolyzed and delignified wood. Two reactor setup schemes were examined for enzyme recycling. One scheme involved a single train of reactors, with the whole pretreated slurry flowing from one reactor to the next, whereas, in the other scheme, the slurry was split among parallel trains of reactors. The activity loss of the enzymes was modeled such that a part of the enzymes entering the reactor lost all their activity. The loss of activity in multiple steps, with enzymes losing only some of their activity, was also modeled. Here the enzymes entering the reactor constituted a mixture with different activities instead of all the enzymes having the same activity like in the previous single step model. Recovering methanol and furfural reduced the minimum ethanol selling price. High temperature ethanol water pretreatment and lignin extraction reduced the minimum ethanol selling price compared to the base case of steam pretreatment followed by alkali extraction. Enzyme recycling also reduces the minimum ethanol selling price. The magnitude of the price reduction depends on the recycling scheme selected and the rate of enzyme deactivation, which has not been measured.

O intestino dos insetos representa uma interface pouco protegida dos agentes externos. A identificação dos mecanismos moleculares envolvidos nos processos fisiológico-digestivos permite encontrar alvos potenciais para o controle de insetos. As moléculas envolvidas na absorção de nutrientes, tamponamento e geração de fluxos de água no intestino médio do inseto-modelo Musca domestica (Diptera Cyclorrhapha) foram identificadas. Para isso, foi feita uma análise da expressão gênica ao longo do intestino médio, a identificação e anotação de proteínas por bioinformática, a confirmação da localização apical das proteínas por análise proteômica de membranas microvilares purificadas e a determinação do papel de algumas das proteínas através de experimentos in vivo utilizando diferentes dietas, corantes e inibidores. A acidificação da região média é consequência da atividade anidrase carbônica que gera prótons que são bombeados por uma H+ V-ATPase apical acompanhados por cloreto transportado por um simporter K+Cl-. O K+ é recuperado por um canal de K+ e a homeostase dos cátions mantida pela Na+/K+-ATPase basolateral. O bicarbonato é eliminado basolateralmente em troca por cloreto por um antiporter. A acidificação é regulada diretamente por um antiporter Na+/H+ e indiretamente por uma proteína envolvida na homeostase do cobre. O muco protetor da região média é tamponado com bicarbonato gerado por uma anidrase carbônica com âncora de GPI e transportado por um antiporter Na+HCO3-/H+Cl-. O excesso de ácido é eliminado por um antiporter Na+/H+ situado na membrana basolateral. A alcalinização da região posterior ocorre pelo transporte apical de NH3 que sequestra os prótons luminais gerando amônio, junto à remoção de prótons em simporte com aminoácidos e peptídeos. A acidificação intracelular, consequência da entrada de aminoácidos com prótons, é regulada por uma H+ V-ATPase basolateral. A geração de fluxos de água é consequência da atividade conjunta de simporters NKCC e KCC ajudados pelas aquaporinas. A inibição dos simporters com inibidores específicos mostrou que o contrafluxo de água está envolvido na reciclagem da enzima tripsina. Por último, o principal lugar de absorção nutrientes no intestino médio é a região posterior, a exceção do cobre que é absorvido na região média. / The gut of insects is a less protected interphase against external agents. The identification of the molecular mechanisms involved in physiological-digestive processes allows one to find potential targets for insect control. The molecules involved in nutrient absorption, buffering and fluid fluxes in the midgut of the insect-model M. domestica (Diptera Cyclorrhapha) were identified. For this, gene expression along the midgut was analyzed; proteins were identified and annotated by bioinformatics; apical localization of proteins was confirmed by proteomics of purified microvillar membranes; the role of proteins was confirmed by in vivo experiments using different diets, dyes and inhibitors. Middle midgut acidification occurs by the action of an apical H+ V-ATPase with protons coming from carbonic anhydrase activity accompanied by chloride transported with potassium by a K+Cl- symporter. Potassium is recovered by a potassium channel, and cation homeostasis maintained by a basolateral Na+/K+-ATPase. Acidification is directly regulated by a Na+/H+ antiporter and indirectly by a copper homeostasis protein. Mucus protecting epithelium is neutralized with bicarbonate generated by a GPI-ancored carbonic anhydrase and transported by a Na+HCO3-/H+Cl- antiporter. Intracellular acidification is avoided by a basolateral Na+/H+ antiporter. Posterior midgut alkalization occurs by the action of an apical ammonia transporter and proton amino acid (and peptide) symporters. Intracellular acid is eliminated by a basolateral H+ V-ATPase. Fluid fluxes are generated by K+Cl- and Na+Cl-Cl- symporters helped by aquaporins. Inhibition of these symporters showed that the countercurrent flux of water allows trypsin recycling. Finally, posterior midgut is the main location of nutrient absorption, except for copper which is absorbed in the middle midgut.