Optimização de pré-tratamento para a hidrólise selectiva da hemicelulose de bagaço de azeitona: estudo da bioconversão dos hidrolisados para a produção de xilitol

Mestrado em Engenharia Alimentar - Instituto Superior de Agronomia

The extracted olive pomace (EOP) is the waste of the olive-pomace oil extraction industry. Currently it is produced in large amounts throughout the entire Mediterranean basin, and there are no effective upgrade technologies for its valorization. As such, it is mainly used as an inexpensive energy source, either on site, or sold to other industries. As pomace oil extraction is carried out in large centralized facilities, this material is highly geographically concentrated, and already located on an industrial site. Furthermore, its significant polysaccharide content (above 45%) as quantified in this work, make it an interesting material for the biotechnological valorization within the biorefinery concept. The hemicellulosic content (that accounts for approximately 50% of all polysaccharides) is particularly relevant and its challenging valorization is studied in this work, by the development of an upgrading strategy targeted to the production of xylitol, a compound with potential applications in the food, pharmaceutical and cosmetic industries. For this, the dilute acid hydrolysis pre-treatment was used for the selective recovery of hemicelluloses. The effect of liquid/solid ratio (LSR) was initially studied, which enabled to define one of the lowest LSR described in literature (3) that enable to get highly concentrated pentose-rich hydrolysates and a low usage of process water. The dilute acid hydrolysis was further optimized by using the Doehlert experimental design to study the effects of time (0 to 180 min) and catalyst (H2SO4) concentration (0.5 to 3.5%) at 130 ºC. The combined severity factor (CS) was used to interpret the results and its limitations are highlighted, so that the use of empirical mathematical models was used instead to fine tune the operational conditions, that were established at 3.5% H2SO4 and 130 min (CS=2.64). Under the optimal conditions it was possible to obtain a hydrolysate containing more than 34 g/L of xylose, one of the highest reported in literature for non-concentrated hydrolysates. Furthermore, glucose was found in relative low amounts (similar to arabinose concentration), rendering this hydrolysate quite interesting for xylose bioconversion. The amounts of potential microbial inhibitors such as aliphatic acids (mainly acetic acid), furans (furfural and hydroxymethylfurfural) and phenolic compounds were high as compared to other hydrolysates. This is a consequence of the high content of extractives (of which soluble phenolic compounds) and the high degree of acetylation of the hemicelluloses and was dealt on the subsequent task, hydrolysate detoxification. The remaining solids have an increased glucan content that can be upgraded into bioethanol, within the integrated biorefinery concept. Hydrolysate detoxification was studied using physico-chemical treatments, specifically neutralization with alkaline agents (NaOH, Ca(OH)2, CaCO3), adsorption to activated charcoal, and evaporation. The best method found was the use of charcoal activated with XIV NaOH (patent pending from this work) that allowed to remove 100% of furfural, 97% hydroxymethylfurfural and 92% of phenolic compounds present in the hydrolysate. In order to access polyols production in BAE hydrolysate, six Debaryomyces hansenii yeast strains both from var. hansenii (CCMI 495, CCMI 470, CCMI 493 and 494) and var. fabryi (CCMI 496 and CCMI 941) were cultivated in chemically defined medium, with both glucose and xylose to mimic hydrolysate composition, in order to have some insight on their pentose metabolism. As expected, they have shown a quite different behavior, with strains belonging to var. fabryi showing a better performance than var. hansenii. Nevertheless, two strains were selected, one from each group, for further studies, The selected strains were CCMI 493 and CCMI 941 that displayed a lower biomass yield, a parameter chosen as indicator that they could potentially dissipate an higher Carbon flux into products. The bioconversion assays were carried out with both strains, in hydrolysate based media, but, conversely to CCMI 941, Debaryomyces hansenii CCMI 493 was not able to grow in these media. For CCMI 941 it was shown that the detoxification step is an absolute requirement, since without it, no growth, or xylitol production, were also observed. Simultaneously, the effect of hydrolysate concentration, aeration and supplementation was also studied through the addition to the hydrolysate of yeast extract, peptone, corn steep liquor and a solution of trace elements, vitamins and minerals (TEVM). The higher biomass and polyols productivity were obtained with the supplementation of low-cost salts (TEVM), what constitutes a significant advantage over other hydrolysates described in the literature.