Evaluation of different glycerol fed-batch strategies in a lab-scale bioreactor for the improved production of a novel engineered β-fructofuranosidase enzyme in Pichia pastoris.

The β-fructofuranosidase enzyme from Aspergillus niger has been extensively used to commercially produce fructooligosaccharides from sucrose. In this study, the native and an engineered version of the β-fructofuranosidase enzyme were expressed in Pichia pastoris under control of the glyceraldehyde-3-phosphate dehydrogenase promoter, and production was evaluated in bioreactors using either dissolved oxygen (DO-stat) or constant feed fed-batch feeding strategies. The DO-stat cultivations produced lower biomass concentrations but this resulted in higher volumetric activity for both strains.

Unlocking peach juice byproduct potential in food waste biorefineries: Phenolic compounds profile, antioxidant capacity and fermentable sugars.

This work assesses an integrated pathway for the revalorization of peach byproduct (PB) within a biorefinery. PB was subjected to an oven-drying (OD) treatment for its evaluation as a storage treatment. It was compared to freeze-drying and untreated material in terms of antioxidant capacity (AOC), phenolic compounds (PC) profile and fermentable sugar production.

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.

Rational engineering of Saccharomyces cerevisiae towards improved tolerance to multiple inhibitors in lignocellulose fermentations.

Background: The fermentation of lignocellulose hydrolysates to ethanol requires robust xylose-capable Saccharomyces cerevisiae strains able to operate in the presence of microbial inhibitory stresses. This study aimed at developing industrial S. cerevisiae strains with enhanced tolerance towards pretreatment-derived microbial inhibitors, by identifying novel gene combinations that confer resistance to multiple inhibitors (thus cumulative inhibitor resistance phenotype) with minimum impact on the xylose fermentation ability.

Synergistic codon optimization and bioreactor cultivation toward enhanced secretion of fungal lignin peroxidase in Pichia pastoris: Enzymatic valorization of technical (industrial) lignins.

Lignin peroxidase (LiP) is a well-recognized enzyme for its ability to oxidize lignins, but its commercial availability is limited, which hinders the biotechnological application of LiP-based bioprocesses in lignocellulose biorefineries. This study evaluated a combination strategy to improve the expression of LiP to promote its practical use. The strategy included optimization of the lipH8 gene of Phanerochaete chrysosporium according to the codon usage of Pichia pastoris, followed by fed-batch fermentation using a 14 L bioreactor (10 L working volume).

Scalable methanol-free production of recombinant glucuronoyl esterase in Pichia pastoris.

Objective: Glucuronoyl esterase (GE) is an emerging enzyme that improves fractionation of lignin-carbohydrate complexes. However, the commercial availability of GE is limited, which hinders the research of GE-based bioprocesses for its industrial application in lignocellulose biorefineries. This study evaluated a workable, cost-effective, and commercially scalable production strategy to improve the ease of GE-based research.

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.

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.

Opuntia ficus-indica cladodes as feedstock for ethanol production by Kluyveromyces marxianus and Saccharomyces cerevisiae.

The feasibility of ethanol production using an enzymatic hydrolysate of pretreated cladodes of Opuntia ficus-indica (prickly pear cactus) as carbohydrate feedstock was investigated, including a comprehensive chemical analysis of the cladode biomass and the effects of limited aeration on the fermentation profiles and sugar utilization. The low xylose and negligible mannose content of the cladode biomass used in this study suggested that the hemicellulose structure of the O. ficus-indica cladode was atypical of hardwood or softwood hemicelluloses.

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.

Application of endo-β-1,4,D-mannanase and cellulase for the release of mannooligosaccharides from steam-pretreated spent coffee ground.

Spent coffee ground (SCG), a present waste stream from instant coffee production, represents a potential feedstock for mannooligosaccharides (MOS) production. MOS can be used in nutraceutical products for humans/animals or added to instant coffee, increasing process yield and improving product health properties. The SCG was evaluated for MOS production by steam pretreatment and enzymatic hydrolysis with a recombinant mannanase and a commercial cellulase cocktail (Acremonium, Bioshigen Co.

Exploring grape marc as trove for new thermotolerant and inhibitor-tolerant Saccharomyces cerevisiae strains for second-generation bioethanol production.

Background: Robust yeasts with high inhibitor, temperature, and osmotic tolerance remain a crucial requirement for the sustainable production of lignocellulosic bioethanol. These stress factors are known to severely hinder culture growth and fermentation performance.

Results: Grape marc was selected as an extreme environment to search for innately robust yeasts because of its limited nutrients, exposure to solar radiation, temperature fluctuations, weak acid and ethanol content.

Enzymatic hydrolysis of spent coffee ground.

Spent coffee ground (SCG) is the main residue generated during the production of instant coffee by thermal water extraction from roasted coffee beans. This waste is composed mainly of polysaccharides such as cellulose and galactomannans that are not solubilised during the extraction process, thus remaining as unextractable, insoluble solids. In this context, the application of an enzyme cocktail (mannanase, endoglucanase, exoglucanase, xylanase and pectinase) with more than one component that acts synergistically with each other is regarded as a promising strategy to solubilise/hydrolyse remaining solids, either to increase the soluble solids yield of instant coffee or for use as raw material in the production of bioethanol and food additives (mannitol).

Enhancing the enzymatic digestibility of sugarcane bagasse through the application of an ionic liquid in combination with an acid catalyst.

Various ionic liquids have been identified as effective pretreatment solvents that can enhance the cellulose digestibility of lignocellulose by removing lignin, one of the main factors contributing to the recalcitrant nature of lignocellulose. 1-Butyl-3-methylimidazolium methylsulfate ([BMiM]MeSO(4)) is a potential delignification reagent, hence its application as a pretreatment solvent for sugarcane bagasse (SB) was investigated. The study also evaluated the benefit of an acid catalyst (i.

Evaluation of steam-treated giant bamboo for production of fermentable sugars.

Giant bamboo plantations are currently being established in the Southern Africa region and can be considered as potential lignocellulosic feedstock for the production of second generation bioethanol. In this study, giant bamboo internodal material was subjected to sulphur dioxide (SO(2)) impregnated steam pretreatment prior to enzymatic hydrolysis. The effect of temperature, residence time, and acidity on the overall sugar recovery and byproduct formation was studied using response surface response technology according to a central composite experimental design (CCD) at a fixed SO(2) concentration of 2.

Effect of different cellulase dosages on cell viability and ethanol production by Kluyveromyces marxianus in SSF processes.

This study was aimed to study the effect of commercial cellulases (Celluclast 1.5 LFG) on Kluyveromyces marxianus CECT 10875 growth and ethanol production in SSF processes. Preliminary tests carried out in glucose (50 g/L) fermentation medium showed that high enzyme amounts (2.

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.

Effect of inhibitors released during steam-explosion pretreatment of barley straw on enzymatic hydrolysis.

The influence of the liquid fraction (prehydrolysate) generated during steam-explosion pretreatment (210 degrees C, 15 min) of barley straw on the enzymatic hydrolysis was determined. Prehydrolysate was analyzed for degradation compounds and sugars' content and used as a medium for enzymatic hydrolysis tests after pH adjusting to 4.8.