Production of chitin-glucan complex by Pichia pastoris

Dissertation for the Degree of Master in BiotechnologyThe yeast Pichia pastoris produces chitin-glucan complex (CGC), as a cell wall component. CGC is composed of two types of biopolymers, chitin and β-glucans, which confer it great potential for use in the food, cosmetic and pharmaceutical industries. CGC hydrolysis, allows obtaining chitin/chitosan and glucans individually. The chitin and chitosan obtained from CGC have the considerable advantage of being of non-animal origin, which further extends their applications. In last year’s, the production of CGC by Pichia pastoris was realized with glycerol as sole carbon source, achieving high cell density. In this study, different substrates were tested for cultivation of P. pastoris and CGC production. In the first part, mixtures of glucose and xylose, in varying proportions, were tested. Since glucose and xylose are two of the main sugar components of lignocellulosic wastes, the ability of P. pastoris to use them as carbon sources would allow their valorization into value-added products. In the second part, several wastes and byproducts generated by different industries were tested for their suitability as substrates for P. pastoris cultivation. In study of the glucose/xylose mixtures, the best performance was achieved in batch bioreactor experiments with 25% xylose in the medium (20 gL-1 of xylose and 60 gL-1 of glucose), where 35.25 gL-1 biomass was obtained in 64 hours of cultivation. CGC content in the cell wall reached 15% with a volumetric productivity of 0.085 gL-1.h-1. The molar ratio of chitin:β-glucan in the extracted biopolymer was 47:53, higher than obtained with crude glycerol (16:84). In the second part of study, several wastes and byproducts (used cooking oil, sugarcane molasses, cheese whey, waste paper and spent coffee grounds) were tested. The results show that P. pastoris presented low biomass concentration using any of these substrates. Nevertheless, in batch bioreactor experiments the best results were achieved with sugarcane molasses, where 17.78 gL-1 biomass were obtained with a CGC content of 17%. Among the tested substrates, the mixtures of glucose/xylose appear to be the most promising due the good CGC production obtained and the high glucosamine molar fraction in produced polymer. This study opens the hypothesis of utilization of lignocellulosic materials with xylose percentages up to 50%