Adaptation of Saccharomyces cerevisiae in a concentrated spent sulphite liquor waste stream for increased inhibitor resistance.

The fermentation of spent sulphite liquor (SSL) from the pulping of hardwoods is limited by the combination of xylose, the primary fermentable sugar and high concentrations of microbial inhibitors that decrease the yeast fermentation ability. The inhibitor resistance phenotypes of xylose-capable Saccharomyces cerevisiae strains were therefore enhanced by combining rational engineering for multi-inhibitor tolerance, with adaptation in concentrated hardwood SSL as selective pressure. The adapted strains were assessed in fermentations with 60-80% v/v concentrated SSL under industrially relevant fermentation conditions. During adaptation, strains produced ethanol concentrations between 11.0 and 15.4 g/L in the range of that reported in literature. The adapted TFA40 and TP50 strains displayed enhanced inhibitor resistance phenotypes and were able to ferment xylose-rich SSL at pH below 5, exhibiting improved ethanol yields relative to the reference strain. Using yeast extract and peptone as nitrogen source in concentrated SSL fermentations further improved ethanol yields. However, strains exhibited a trade-off between resistance and ethanol productivity, indicating a carbon/energy cost for the expression of this inhibitor tolerance phenotype. KEY POINTS : • Achieved fermentation of xylose-rich hardwood spent sulphite liquor at pH below 5.0 • Adaptation of xylose-capable S. cerevisiae in concentrated spent sulphite liquor • Adapted strains exhibited enhanced inhibitor resistance phenotypes.

Lignin enrichment and enzyme deactivation as the root cause of enzymatic hydrolysis slowdown of steam pretreated sugarcane bagasse.

The enzymatic hydrolysis (EH) rate normally decreases during the hydrolysis, leaving unhydrolyzed material as residue. This phenomenon occurs during the hydrolysis of both cellulose (avicel) and lignocellulosic material, in nature or even pretreated. The progression of EH of steam pretreated sugarcane bagasse was associated with an initial (fast), intermediate (slower) and recalcitrant (slowest) phases, at glucan to glucose conversion yields of 61.7, 81.6 and 86%, respectively. Even though the EH of avicel as a simpler material than steam pretreated sugarcane bagasse, EH slowdown was present. The less thermo-stable endo-xylanase lost 58% of initial enzyme activity, followed by ß-glucosidase that lost 16%, culminating in FPase activity loss of 30% in the first 24hours. After 72hours of EH the total loss of FPase activity was 40% compared to the initial activity. Analysis of the solid residue from EH showed that lignin content, phenolic compounds and ash increased while glucan decreased as hydrolysis progressed. During the initial fast phase of EH, the total solid residue surface area consisted predominantly of internal surface area. Thereafter, in the intermediate and recalcitrant phases of EH, the ratio of external:internal surface area increased. The proposed fiber damage and decrease in internal surface area, probably by EH action, was visualized by scanning electron microscopy imagery. The higher lignin/glucan ratio as EH progressed and enzyme deactivation by thermo instability were the main effects observed, respectively to substrate and enzyme.

Steam explosion pretreatment of triticale (× Triticosecale Wittmack) straw for sugar production.

Triticale, a non-food based, low-cost and well-adapted crop in marginal lands has been considered as a potential 1G and 2G feedstock for bio-ethanol production. In this work, triticale straw was evaluated as a source of fermentable sugars by combination of uncatalyzed steam explosion and enzymatic hydrolysis. Pretreatment conditions with severities from 3.05 to 4.12 were compared in order to identify conditions that favour the recovery of hemicellulose-derived sugars, cellulose digestibility or the combined sugars yield (CSY) from the pretreatment-enzymatic hydrolysis. Xylose oligosaccharide was the major sugar in hydrolysates from all pretreatment conditions. Maximum hemicellulose-sugars recovery (52% of the feedstock content) was obtained at 200 °C and 5 min. The highest cellulose digestibility (95%) was found at 200 °C - 15 min, although glucose recovery from hydrolysis was maximised at 200 °C - 10 min (digestibility >92%) due to higher mass yield of pretreated solids. The maximum CSY (nearly 77% of theoretical content) was obtained at 200 °C - 5 min. Sugar loss after pretreatment was observed to higher extent at harsher severities. However, the concentrations of sugar degradation products and acetic acid were at levels below tolerance limits of the downstream biological conversions. Steam explosion pretreatment without acid impregnation is a good technology for production of fermentable sugars from triticale straw. This work provides foundation for future autohydrolysis steam explosion optimization studies to enhanced sugars recovery and digestibility of triticale straw.

Impact of cultivar selection and process optimization on ethanol yield from different varieties of sugarcane.

BACKGROUND: The development of 'energycane' varieties of sugarcane is underway, targeting the use of both sugar juice and bagasse for ethanol production. The current study evaluated a selection of such 'energycane' cultivars for the combined ethanol yields from juice and bagasse, by optimization of dilute acid pretreatment optimization of bagasse for sugar yields. METHOD: A central composite design under response surface methodology was used to investigate the effects of dilute acid pretreatment parameters followed by enzymatic hydrolysis on the combined sugar yield of bagasse samples. The pressed slurry generated from optimum pretreatment conditions (maximum combined sugar yield) was used as the substrate during batch and fed-batch simultaneous saccharification and fermentation (SSF) processes at different solid loadings and enzyme dosages, aiming to reach an ethanol concentration of at least 40 g/L. RESULTS: Significant variations were observed in sugar yields (xylose, glucose and combined sugar yield) from pretreatment-hydrolysis of bagasse from different cultivars of sugarcane. Up to 33% difference in combined sugar yield between best performing varieties and industrial bagasse was observed at optimal pretreatment-hydrolysis conditions. Significant improvement in overall ethanol yield after SSF of the pretreated bagasse was also observed from the best performing varieties (84.5 to 85.6%) compared to industrial bagasse (74.5%). The ethanol concentration showed inverse correlation with lignin content and the ratio of xylose to arabinose, but it showed positive correlation with glucose yield from pretreatment-hydrolysis. The overall assessment of the cultivars showed greater improvement in the final ethanol concentration (26.9 to 33.9%) and combined ethanol yields per hectare (83 to 94%) for the best performing varieties with respect to industrial sugarcane. CONCLUSIONS: These results suggest that the selection of sugarcane variety to optimize ethanol production from bagasse can be achieved without adversely affecting juice ethanol and cane yield, thus maintaining first generation ethanol production levels while maximizing second generation ethanol production.

Xylanase contribution to the efficiency of cellulose enzymatic hydrolysis of barley straw.

In this study, different enzyme preparations available from Novozymes were assessed for their efficiency to hydrolyze lignocellulosic materials. The enzyme mixture was evaluated on a pretreated cellulose-rich material, and steam-exploded barley straw pretreated under different temperatures (190, 200, and 210 degrees C, respectively) in order to produce fermentable sugars. Results show that xylanase supplementation improves initial cellulose hydrolysis effectiveness of water-insoluble solid fraction from all steam-exploded barley straw samples, regardless of the xylan content of substrate. The mixture constituted by cellulase: beta-glucosidase: endoxylanase of the new kit for lignocellulose conversion at a ratio 10:1:5% ([v/w], enzyme [E]/substrate [S]) provides the highest increment of cellulose conversion in barley straw pretreated at 210 degrees C, for 10 min.