Targeting to improve the utilization of lignocellulosic residues in the ethanol processing industry, this work aimed to test if the product inhibition of the enzymatic hydrolysis could be relieved by extractive reaction using aqueous two-phase systems (ATPS). The performance of enzymatic hydrolysis in ATPS is not well defined in literature. In this thesis, this extractive reaction was tested in terms of experimental conversion of sugarcane bagasse, simulations through conceptual process design and economic feasibility. A thermodynamic framework was developed in order to predict ATPS formation. The screening of ATPS and partition coefficient of the solutes were performed in a high throughput station. The ATPS were composed by polymer and salt. The enzymes were represented by the enzymatic cocktail Cellic CTec (Novozymes). The development of this platform consisted of two main parts: determination of phase diagrams (binodal curves and tie lines) and quantification of the solutes (sugar and proteins) in both top and bottom phases. The most promising ATPS were experimentally explored for enzymatic hydrolysis of sugarcane bagasse. Process design simulated two scenarios: hydrolysis occurring in the bottom phase and in the top phase. Topics such as the adsorption of phase forming components to the bagasse fibers and the influence of enzyme load on the hydrolysis were explored. The sugarcane bagasse hydrolysis in ATPS was conceptually assessed through the implementation of a model composed by two parts: hydrolysis and ATPS multi-batch separation. The designed case characterized by the ATPS hydrolysis was compared to the base case defined as conventional hydrolysis. Regarding the thermodynamic modelling of ATPS, the application of Flory-Huggins (FH) model to predict phase separation in polymer-salt systems was assessed. The implementation and analysis of FH theory involved the estimation of interchange energy (푤푖푗) and the calculation of phase diagrams. There were no statistical differences in determining the phase diagram in HTP platforms and bench-scale, verifying the reliability of methods and equipment suggested in this work. Moreover, tailored approaches to quantify the solutes were 8 presented, taking into account the limitations of techniques that can be applied with ATPS due to the interference of phase forming components with the analytics. This fast methodology proposes to screen up to six different polymer-salt systems in eight days and supplies the results to understand the influence of sugar and protein concentrations on their partition coefficients. Exploring experimentally the ATPS hydrolysis provided strategies on how to conduct extractive enzymatic hydrolysis in ATPS and how to explore the experimental results in order to design a feasible process. In the conceptual design of extractive enzymatic hydrolysis, one of the major bottlenecks identified was the partitioning of glucose to both phases. The resultant conceptual process design operates as a tool to evaluate ATPS hydrolysis and compare it to conventional one. On the other hand, the thermodynamic model could not quantitatively describe the data. This occurs mainly because of the strong influence of random experimental errors on the estimation of interchange energy, systematic errors when translating the observed data to calculated partition concentrations, and FH not being an exact description of phase separation in salt based ATPS. The high throughput screening methodology indicated ATPS able to partition sugar and enzymes. The selected ATPS presented no significant improvements to perform the enzymatic conversion of sugarcane bagasse compared to the conventional hydrolysis. The main reasons were the influence of phase forming components on the enzymatic activity and the low selectivity of sugars in the ATPS. To disclose the application of ATPS in the ethanol processing industry, the recovery and reuse of the phase forming components are imperative for economic feasibility. Moreover, the developed high throughput platform could be further employed to exhaustively screen systems to design effective ATPS for the partition of sugars and proteins in polymer-salt systems.

Trichoderma harzianum P49P11 was selected among several other microorganisms at the Biorenewables National Laboratory (LNBR, Brazil) and it was considered a promising strain to produce cellulase. Here in this project, a mathematical model and simulation platforms were developed as potential tools to be used for cellulase maximization using fed-batch mode (Chapter 2). Feeding strategies were simulated to maximize cellulase production, at first, only using cellulose as the substrate, and then using glycerol for cell growth and cellulose for cellulase production. Although the mathematical model and simulation platforms were built up for a wild type strain, these tools help to predict data and they can be adapted for optimized strains.
Chapter 3 evaluates the continuous production of enzymes using different carbon sources under carbon-limited conditions. It was found that glucose has a positive influence on the production of enzymes that can catalyse the hydrolysis of p-nitrophenyl-β-D-glucopyranoside (PNPGase). Sucrose and fructose seem to inhibit PNPGase synthesis; however, these substrates could also have a positive influence on the synthesis of other enzymes not evaluated in this project. Cells can uptake glucose without the need to synthesize extracellular enzymes like PNPGase. The increase in the production of PNPGase during the continuous culture using glucose as the carbon source indicates the presence of inducers. It was also discovered in this project that polysaccharides were present in the supernatant of all conditions using glucose, fructose/glucose and sucrose (Chapter 4 and Chapter 5). This suggests that the possible inducers could have come from fragments of the extracellular polysaccharides.
Sugar analysis showed the presence of sugar with the same retention time as gentiobiose in the supernatant of the conditions using glucose as the carbon source, which could be a fragment from polymers released from the cell wall. Gentiobiose could be acting as an inducer of enzymes. In addition, a mechanism was also proposed for continuous PNPGase production under glucose-limited conditions assuming that PNPGase includes beta-glucosidase (Chapter 4).
The carbon sources used under carbon-limited conditions influenced the PNPGase productivity and possibly the whole enzymatic cocktail secreted by the fungus. For this reason, shotgun proteomics and SDS-PAGE analysis were performed for the proteins present in the supernatant of the conditions using glucose, fructose/glucose and sucrose (Chapter 4). The shotgun proteomics analysis suggested that the different carbon sources used provided the production of different extracellular proteins including several uncharacterized proteins, which can also include different enzymes. This brings the possibility of creating a hypothesis that different carbon sources easily assimilated by the cells could lead to the synthesis of different inducers (fragments of extracellular polysaccharides), which could induce the synthesis of different enzymes under carbon-limited conditions.
Extracellular polysaccharides were the by-products discovered in this project during the production of enzymes under carbon-limited conditions. The behaviour of intracellular metabolites (glycolysis, citric acid cycle, pentose phosphate pathway and nucleotides) was evaluated under four different conditions in duplicate during the production of extracellular polysaccharides by Trichoderma harzianum under carbon-limited conditions (Chapter 5). This chapter has provided the first step for the optimization of the production of extracellular polysaccharides and the information about the behaviour of intracellular metabolites using this wild type strain is essential to the development of optimal strains.