Aqueous two-phase micellar systems (ATPMS) can be exploited in separation science for the extraction/purification of desired biomolecules. Prior to phase separation the surfactant solution reaches a cloud point temperature, which is influenced by the presence of electrolytes. In this work, we provide an investigation on the cloud point behavior of the nonionic surfactant C10E4 in the presence of NaCl, Li2SO4 and KI. We also investigated the salts' influence on a model protein partitioning. NaCl and Li2SO4 promoted a depression of the cloud point. The order of salts and the concentration that decreased the cloud point was: Li2SO4 0.5 M > NaCl 0.5 M ≈ Li2SO4 0.2 M. On the other hand, 0.5 M KI dislocated the curve to higher cloud point values. For our model protein, glucose-6-phosphate dehydrogenase (G6PD), partitioning experiments with 0.5 M NaCl or 0.2 M Li2SO4 at 13.85 ºC showed similar results, with K G6PD ~ 0.46. The lowest partition coefficient was obtained in the presence of 0.5 M KI (K G6PD = 0.12), with major recovery of the enzyme in the micelle-dilute phase (%Recovery = 90%). Our results show that choosing the correct salt to add to ATPMS may be useful to attain the desired partitioning conditions at more extreme temperatures. Furthermore, this system can be effective to separate a target biomolecule from fermented broth contaminants.

The actual biotechnology industry demands fast and economic upstream and downstream processes to purify biomolecules. In this context, different purification techniques, that offer both high recovery and purity to the final product, have been assayed by different research groups. Liquid-liquid extraction with aqueous two-phase systems is one of the most studied methodologies for bio-separation. This technique presents several advantages such as mild conditions of working, cost-effectiveness, short-time consumption and high recovery percentage of the final product. With the aim to present a comparison of liquid-liquid extractions with other techniques, several aqueous two-phase extraction processes of biomolecules are presented in this review. We presented the advantages and disadvantages of them as of the compared systems. In general, the highest final product purities are achieved when different methodologies are combine, being the chromatographic ones the most applied in the last stages for the high purification factor obtained after them. Alternative methodologies, such as aqueous two-phase systems (ATPS), i.e., PEG/salts or ionic liquids; aqueous two-phase micellar systems, using solvents and surfactants; and extractive fermentation with ATPS, are relevant for both cost-effectiveness and time-saving of the purification process.

La industria biotecnológica actual exige procesos rápidos y económicos para la producción y la purificación de biomoléculas. En este contexto, diferentes técnicas separativas que ofrezcan un alto rendimiento y una alta pureza del producto final han sido evaluadas por diferentes grupos de investigación. La extracción líquido-líquido con sistemas bifásicos acuosos (SBAs) es una de las metodologías más estudiadas para bio-separación. Esta técnica presenta varias ventajas, tales como condiciones suaves de trabajo, alta relación costo-beneficio, cortos tiempos de consumo y alto porcentaje de recuperación del producto final. Con el objetivo de comparar extracciones líquido-líquido con otras técnicas de separación, diferentes procesos de extracción de biomoléculas serán presentados en este trabajo de revisión. Adicionalmente, se presentarán las ventajas y desventajas de cada uno de ellos. En general, los más altos grados de pureza del producto final fueron obtenidos al combinar diferentes metodologías, siendo las cromatográficas las más aplicados en las últimas etapas debido a los elevados factores de purificación obtenidos después de ellas. Metodologías alternativas como por ejemplo, SBAs basados en mezclas de PEG/sal o líquidos iónicos/sal, sistemas micelares de dos fases acuosas (SMDFAs) formados por solventes o surfactantes; fermentación extractiva utilizando SBA, también resultan ser relevantes debido a sus bajos costos y cortos tiempo en sus procesos de purificación.

Xylitol has sweetening, anticariogenic and clinical properties that have attracted the attention of the food and pharmaceutical industries. The conversion of sugars from lignocellulosic biomass into xylitol by D-xylose-fermenting yeast represents an alternative to the chemical process for producing this polyol. A good source of D-xylose is sugarcane bagasse, which can be hydrolyzed with dilute acid. However, acetic acid, which is toxic to the yeast, also appears in the hydrolysate, inhibiting microbe metabolism. Xylitol production depends on the initial D-xylose concentration, which can be increased by concentrating the hydrolysate by vacuum evaporation. However, with this procedure the amount of acetic acid is also increased, aggravating the problem of cell inhibition. Hydrolysate treatment with powdered activated charcoal is used to remove or decrease the concentration of this inhibitor, improving xylitol productivity as a consequence. Our work was an attempt to improve the fermentation of Candida guilliermondii yeast in sugarcane bagasse hydrolysate by treating the medium with seven types of commercial powdered activated charcoals (Synth, Carbon Delta A, Carbon Delta G, Carbon 117, Carbon 118L, Carbon 147 and Carvorite), each with its own unique physicochemical properties. Various adsorption conditions were established for the variables temperature, contact time, shaking, pH and charcoal concentration. The experiments were based on multivariate statistical concepts, with the application of fractional factorial design techniques to identify the variables that are important in the process. Subsequently, the levels of these variables were quantified by overlaying the level curves, which permitted the establishment of the best adsorption conditions for attaining high levels of xylitol volumetric productivity and D-xylose-to-xylitol conversion. This procedure consisted in increasing the original pH of the hydrolysate to 7.0 with CaO and reducing it to 5.5 with H3PO4. Next, the hydrolysate was treated under adsorption conditions employing CDA powdered activated charcoal (1%) for 30 min at 60ºC, 100 rpm and pH 2.5. The optimized xylitol volumetric productivity (0.50 g/L h) corresponded to a D-xylose-to-xylitol conversion of 0.66 g/g.