Improved sugar co-utilisation by encapsulation of a recombinant Saccharomyces cerevisiae strain in alginate-chitosan capsules

Abstract

BackgroundTwo major hurdles for successful production of second-generation bioethanol are the presence of inhibitory compounds in lignocellulosic media, and the fact that Saccharomyces cerevisiae cannot naturally utilise pentoses. There are recombinant yeast strains that address both of these issues, but co-utilisation of glucose and xylose is still an issue that needs to be resolved. A non-recombinant way to increase yeast tolerance to hydrolysates is by encapsulation of the yeast. This can be explained by concentration gradients occuring in the cell pellet inside the capsule. In the current study, we hypothesised that encapsulation might also lead to improved simultaneous utilisation of hexoses and pentoses because of such sugar concentration gradients.ResultsIn silico simulations of encapsulated yeast showed that the presence of concentration gradients of inhibitors can explain the improved inhibitor tolerance of encapsulated yeast. Simulations also showed pronounced concentration gradients of sugars, which resulted in simultaneous xylose and glucose consumption and a steady state xylose consumption rate up to 220-fold higher than that found in suspension culture. To validate the results experimentally, a xylose-utilising S. cerevisiae strain, CEN.PK XXX, was constructed and encapsulated in semi-permeable alginate-chitosan liquid core gel capsules. In defined media, encapsulation not only increased the tolerance of the yeast to inhibitors, but also promoted simultaneous utilisation of glucose and xylose. Encapsulation of the yeast resulted in consumption of at least 50% more xylose compared with suspended cells over 96-hour fermentations in medium containing both sugars. The higher consumption of xylose led to final ethanol titres that were approximately 15% higher. In an inhibitory dilute acid spruce hydrolysate, freely suspended yeast cells consumed the sugars in a sequential manner after a long lag phase, whereas no lag phase was observed for the encapsulated yeast, and glucose, mannose, galactose and xylose were utilised in parallel from the beginning of the cultivation.ConclusionsEncapsulation of xylose-fermenting S. cerevisiae leads to improved simultaneous and efficient utilisation of several sugars, which are utilised sequentially by suspended cells. The greatest improvement is obtained in inhibitory media. These findings show that encapsulation is a promising option for production of second-generation bioethanol.

Flocculation Causes Inhibitor Tolerance in Saccharomyces cerevisiae for Second-Generation Bioethanol Production

“…5D and Table 3). Similar effects have recently been shown by encapsulation of a xylose-fermenting yeast, which led to improved simultaneous consumption of glucose, mannose, galactose, and xylose (19). This can be explained by the same reasoning as for the increased inhibitor tolerance.…”

“…In that study, it was concluded that the tightly packed cell community created by the encapsulation of the cells increases the overall robustness of the yeast by letting cells close to the membrane convert inhibitors, allowing inner lying cells to ferment sugars because of lower inhibitor concentrations. By computer simulations, it has been shown that diffusion limitations in the encapsulated cell pellet lead to concentration gradients of both nutrients and convertible inhibitors (19). It has also been shown that encapsulation of yeast induces a starvation stress response which increases the robustness of the yeast community (11,17).…”

“…As an extension of this, it has also been shown that the simultaneous glucose and xylose utilization by a recombinant S. cerevisiae was improved by encapsulation (19). Glucose is presumably consumed by cells close to the membrane, while xylose is consumed by cells closer to the core (19). With this reasoning, the protective effect does not mainly come from the membrane of the capsule, and hypothetically it would be observed also if the cells were kept tightly together by other means.…”

“…Second, it was shown that conversion of the inhibiting compounds is necessary for the increased robustness, and a model was proposed in which the cells close to the capsule membrane convert inhibitors, leaving subinhibitory levels for inner lying cells (11). As an extension of this, it has also been shown that the simultaneous glucose and xylose utilization by a recombinant S. cerevisiae was improved by encapsulation (19). Glucose is presumably consumed by cells close to the membrane, while xylose is consumed by cells closer to the core (19).…”

Matrices (re)loaded: Durability, viability, and fermentative capacity of yeast encapsulated in beads of different composition during long‐term fed‐batch culture

“…111,112 Overall, heat-shock resistance in these matrices was similar to that previously reported for alginate-encapsulated S. cerevisiae. 28,113,114 Upon encapsulation and upon entry into stationary phase, yeast ceases to divide, which is accompanied by thickening of their cell wall and accumulation of reserve carbohydrates, both of which help to protect cells from stress. 28,113,114 Upon encapsulation and upon entry into stationary phase, yeast ceases to divide, which is accompanied by thickening of their cell wall and accumulation of reserve carbohydrates, both of which help to protect cells from stress.…”