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Xylooligomers are strong inhibitors of cellulose hydrolysis by enzymes

Typically, the enzymatic hydrolysis rate of lignocellulosic biomass is fast initially but then slows down more rapidly than can be explained by just consumption of substrate. Although several factors including enzyme inhibition, enzyme deactivation, a drop in substrate reactivity, or nonproductive binding of enzyme to lignin could be responsible for this loss of effectiveness, we recently reported evidence that xylose, xylan, and xylooligomers dramatically decrease conversion rates and yields, but clarification was still needed for the magnitude of their effect. Therefore, in this study, xylan and various xylooligomers were added to Avicel hydrolysis at low enzyme loadings and found to have a greater effect than adding equal amounts of xylose derived from these materials or when added separately. Furthermore, xylooligomers were more inhibitory than xylan or xylose in terms of a decreased initial hydrolysis rate and a lower final glucose yield even for a low concentration of 1.67 mg/ml. At a higher concentration of 12.5mg/ml, xylooligomers lowered initial hydrolysis rates of Avicel by 82% and the final hydrolysis yield by 38%. Mixed DP xylooligomers showed strong inhibition on cellulase enzymes but not on beta-glucosidase enzymes. By tracking the profile change of xylooligomers, a large portion of the xylooligomers was found to be hydrolyzed by Spezyme CP enzyme preparations, indicating competitive inhibition by mixed xylooligomers. A comparison among glucose sugars and xylose sugars also showed that xylooligomers were more powerful inhibitors than well-established glucose and cellobiose.

Supplementation with xylanase and β-xylosidase to reduce xylo-oligomer and xylan inhibition of enzymatic hydrolysis of cellulose and pretreated corn stover

BackgroundHemicellulose is often credited with being one of the important physical barriers to enzymatic hydrolysis of cellulose, and acts by blocking enzyme access to the cellulose surface. In addition, our recent research has suggested that hemicelluloses, particularly in the form of xylan and its oligomers, can more strongly inhibit cellulase activity than do glucose and cellobiose. Removal of hemicelluloses or elimination of their negative effects can therefore become especially pivotal to achieving higher cellulose conversion with lower enzyme doses.ResultsIn this study, cellulase was supplemented with xylanase and β-xylosidase to boost conversion of both cellulose and hemicellulose in pretreated biomass through conversion of xylan and xylo-oligomers to the less inhibitory xylose. Although addition of xylanase and β-xylosidase did not necessarily enhance Avicel hydrolysis, glucan conversions increased by 27% and 8% for corn stover pretreated with ammonia fiber expansion (AFEX) and dilute acid, respectively. In addition, adding hemicellulase several hours before adding cellulase was more beneficial than later addition, possibly as a result of a higher adsorption affinity of cellulase and xylanase to xylan than glucan.ConclusionsThis key finding elucidates a possible mechanism for cellulase inhibition by xylan and xylo-oligomers and emphasizes the need to optimize the enzyme formulation for each pretreated substrate. More research is needed to identify advanced enzyme systems designed to hydrolyze different substrates with maximum overall enzyme efficacy.

Impact of surfactants on pretreatment of corn stover

Lignin in pretreated cellulosic biomass can non-productively adsorb cellulase, resulting in loss of a significant portion of this expensive protein. In addition, lignin interferes with the path for cellulase action, slowing down hydrolysis. Thus, the effectiveness of enzymatic hydrolysis of pretreated lignocellulosic biomass can be significantly enhanced if lignin is removed or effectively modified before adding enzymes. In this study, the enzymatic digestibilities of solids resulting from using the surfactants Tween-80, dodecylbenzene sulfonic acid, and polyethylene glycol 4000 during water-only or dilute acid pretreatment of corn stover at 140-220 degrees C were evaluated. All of these surfactants increased lignin removal during pretreatment and reduced non-productive binding of enzymes on the biomass surface, but Tween-80 increased enzymatic hydrolysis yields and enhanced total sugar recovery more than the other two. Surfactant pretreatment was found to improve lignin solubility, which could improve cellulose digestibility by reducing unproductive binding to enzyme, and also appeared to enhance performance by modifying the biomass surface.

Hydrolysis of different chain length xylooliogmers by cellulase and hemicellulase.

Commercial cellulase complexes produced by cellulolytic fungi contain enzyme activities that are capable of hydrolyzing non-cellulosic polysaccharides in biomass, primarily hemicellulose and pectins, in addition to cellulose. However, xylanase activities detected in most commercial enzyme preparations have been shown to be insufficient to completely hydrolyze xylan, resulting in high xylooligomer concentrations remaining in the hydrolysis broth. Our recent research showed that these xylooligomers are stronger inhibitors of cellulase activity than others have previously established for glucose and cellobiose, making their removal of great importance. In this study, a HPLC system that can measure xylooligomers with degrees of polymerization (DP) up to 30 was applied to assess how Spezyme CP cellulase, Novozyme 188 β-glucosidase, Multifect xylanase, and non-commercial β-xylosidase enzymes hydrolyze different chain length xylooligomers derived from birchwood xylan. Spezyme CP cellulase and Multifect xylanase partially hydrolyzed high DP xylooligomers to lower DP species and monomeric xylose, while β-xylosidase showed the strongest ability to degrade both high and low DP xylooligomers. However, about 10-30% of the higher DP xylooligomers were difficult to be breakdown by cellulase or xylanase and about 5% of low DP xylooligomers (mainly xylobiose) proved resistant to hydrolysis by cellulase or β-glucosidase, possibly due to low β-xylosidase activity in these enzymes and/or the precipitation of high DP xylooligomers.

Enhancement of enzymatic saccharification of corn stover with sequential Fenton pretreatment and dilute NaOH extraction

In this study, an effective method by the sequential Fenton pretreatment and dilute NaOH extraction (FT-AE) was chosen for pretreating corn stover. Before dilute NaOH (0.75 wt%) extraction at 90 °C for 1h, Fenton reagent (0.95 g/L of FeSO4 and 29.8 g/L of H2O2) was employed to pretreat CS at a solid/liquid ratio of 1/20 (w/w) at 35 °C for 30 min. The changes in the cellulose structural characteristics (porosity, morphology, and crystallinity) of the pretreated solid residue were correlated with the enhancement of enzymatic saccharification. After being enzymatically hydrolyzed for 72 h, the reducing sugars and glucose from the hydrolysis of 60 g/L FT-AE-CS pretreated could be obtained at 40.96 and 23.61 g/L, respectively. Finally, the recovered hydrolyzates containing glucose had no inhibitory effects on the ethanol fermenting microorganism. In conclusion, the sequential Fenton pretreatment and dilute NaOH extraction has high potential application in future.

Improving enzymatic saccharification of bamboo shoot shell by alkalic salt pretreatment with H2O2

Pretreatment of bamboo shoot shell (BSS) by a combination of alkalic salts with hydrogen peroxide (H2O2) was evaluated for its delignification effect and for its ability to enhance enzymatic saccharification of pretreated solids. By comparing different alkalic salts, the combination of 9% Na3PO4·12H2O and 0.3g/g H2O2 (ASHP) was identified as an effective system that showed the highest delignification of 87.7% and the total reducing sugar yield of 97.1% when pretreated BSS at a solid to liquid ratio of 1/20 (w/w) at 80°C for 2h. The delignification effect and the disruption of the lignocelluloses structure by this novel pretreatment method were deduced to be the main reasons that led to enhanced enzymatic saccharification as supported by the chemical composition analysis and the results of SEM, FTIR and XRD analyses of the untreated and alkalic salt pretreated BSS.

A combined sodium phosphate and sodium sulfide pretreatment for enhanced enzymatic digestibility and delignification of corn stover

Na3PO4 and Na2S were employed as efficient alkaline catalysts for the pretreatment of corn stover. To systematically obtain optimal conditions, the effects of critical pretreatment parameters including sodium phosphate concentration (1-4%), sulfidity (0-20%), pretreatment temperature (100-120°C), and reaction time (20-60min) on the reducing sugar yield of pretreated substrates were evaluated in a lab-scale using the response surface methodology. Pretreated under the sodium phosphate concentration of 4%, sulfidity of 10%, temperature of 120°C, and reaction time of 40min, the reducing sugar yield and glucose yield of the pretreated corn stover achieved 91.11% and 64.01%, respectively, with a moderate enzyme loading of 30FPU/g substrate. Additionally, a strong correlation (R(2)=0.971 and R(2)=0.954) between the delignification and the reducing sugar yield (or glucose yield) was observed by this pretreatment method. These results evidently support that the combined Na3PO4-Na2S pretreatment is an effective and feasible method for processing lignocellulosic biomass.

Catalytic conversion of corncob and corncob pretreatment hydrolysate to furfural in a biphasic system with addition of sodium chloride

Catalytic conversion of corncob pretreatment hydrolysate and raw corncob into furfural in a modified biphasic system by SO42-/SnO2- MMT solid catalyst has been developed. The influence of the organic solvent type, organic to water phase ratio, sodium chloride concentration, reaction temperature and time on the furfural production were comparatively evaluated. The results showed that furfural yields of 81.7% and 66.1% were achieved at 190°C for 15mins and 190°C for 20mins, respectively, for corncob pretreatment hydrolysate and raw corncob by this solid catalyst. The solid catalyst used in this study exhibited good stability and high efficiency applied in the modified biphasic system in addition to excellent recyclability. The proposed catalytic system displayed high performance for catalytic conversion of lignocellulosic biomass into important platform chemicals and has great potential in industrial application.

One-pot chemo-enzymatic synthesis of furfuralcohol from xylose

Furfuralcohol (FOL) is an important intermediate for the production of lysine, ascorbic acid, and lubricants. It can be used as a hypergolic fuel in rocketry. In this study, it was attempted to synthesize FOL from xylose by tandem catalysis with solid acid SO42-/SnO2-Montmorillonite and recombination Escherichia coli CCZU-K14 whole cells. Using SO42-/SnO2-Montmorillonite (3.0wt% dosage) as catalyst, a highest furfural yield of 41.9% was achieved from xylose at 170°C for 20min. Furthermore, Escherichia coli CCZU-K14 whole cells were used for bioconverting furfural to FOL. The optimum biocatalytic reaction temperature, reaction pH, cosubstrate concentration, and substrate concentration were 30°C, 6.5, 1.5mol glucose/mol furfural, and 200mM, respectively. Finally, the yield of FOL from 200mM furfural was achieved to 100% by Escherichia coli CCZU-K14 whole cells after 24h. In conclusion, this strategy show high potential application for the effective synthesis of FOL.

Mild alkaline presoaking and organosolv pretreatment of corn stover and their impacts on corn stover composition, structure, and digestibility

An efficient strategy was developed in current work for biochemical conversion of carbohydrates of corn stover into monosaccharides. Corn stover was first presoaked in mild alkaline solution (1% Na2S) under 40°C for 4h, after which about 35.3% of the lignin was successfully removed while the specific surface area was notably enlarged. Then the presoaked solids were subjected to organosolv pretreatment that employed 20% methanol with an addition of 0.2% HCl as catalyst at 160°C for 20min, and the maximum total sugar yield of the pretreated corn stover achieved was 98.6%. The intact structure of corn stover was disrupted by this two-step process, which resulted in a porous but crystalline structure of the regenerated solids that were mainly composed of cellulose. The enlarged specific surface area and increased accessibility made the regenerated solids highly digestible by a moderate enzyme loading.