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.@en

This article provides an analysis on published models used to calculate phase separation in aqueous two-phase systems (ATPS) based on Flory-Huggins (FH) theory, in terms of problem formulation and mathematical solving algorithm. An integrated algorithm is presented, showing different mathematical approaches of using the FH theory. The algorithm involves the estimation of interchange energy, and the calculation of phase compositions. Based on experimental data, the thermodynamic model can provide a useful framework to perform a sensitivity analysis on parameters, in order to understand the influence of salt type, polymer molecular mass, and ionic strength on phase separation. However, this model, restricted to entropic and enthalpic terms, cannot quantitatively describe the data. This occurs mainly because of the strong influence of random experimental errors on the estimation of interchange energy and FH not being an exact description of phase separation in salt based ATPS. After providing a literature overview and mathematical analysis, we bring to the field the application of the FH theory for selecting ATPS and its limitations. @en

The enzymatic conversion of lignocellulosic material to sugars can provide a carbon source for the production of energy (fuels) and a wide range of renewable products. However, the efficiency of this conversion is impaired due to product (sugar) inhibition. Even though several studies investigate how to overcome this challenge, concepts on the process to conduct the hydrolysis are still scarce in literature. Aqueous two-phase systems (ATPS) can be applied to design an extractive reaction due to their capacity to partition solutes to different phases in such a system. This work presents strategies on how to conduct extractive enzymatic hydrolysis in ATPS and how to explore the experimental results in order to design a feasible process. While only a limited number of ATPS was explored, the methods and strategies described could easily be applied to any further ATPS to be explored. We studied two promising ATPS as a subset of a previously high throughput screened large set of ATPS, providing two configurations of processes having the reaction in either the top phase or in the bottom phase. Enzymatic hydrolysis in these ATPS was performed to evaluate the partitioning of the substrate and the influence of solute partitioning on conversion. Because ATPS are able to partition inhibitors (sugar) between the phases, the conversion rate can be maintained. However, phase forming components should be selected to preserve the enzymatic activity. The experimental results presented here contribute to a feasible ATPS-based conceptual process design for the enzymatic conversion of lignocellulosic material.

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Aqueous two-phase systems (ATPS) can be applied to enzymatic reactions that are affected by product inhibition. In the biorefinery context, sugars inhibit the cellulolytic enzymes in charge of converting the biomass. Here, we present a strategy to select an ATPS (formed by polymer and salt) that can separate sugar and enzymes. This automated and miniaturized method is able to determine phase diagrams and partition coefficients of solutes in these. Tailored approaches to quantify the solutes are 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. The developed high-throughput (HT) platform identifies suitable phase forming components and the tie line of operation. 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.

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