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BackgroundWe recently confirmed that the deactivation of T. reesei cellulases at the air-liquid interface reduces microcrystalline cellulose conversion at low enzyme loadings in shaken flasks. It is one of the main causes for lowering of cellulose conversions at low enzyme loadings. However, supplementing cellulases with small quantities of surface-active additives in shaken flasks can increase cellulose conversions at low enzyme loadings. It was also shown that cellulose conversions at low enzyme loadings can be increased in unshaken flasks if the reactions are carried for a longer time. This study further explores these recent findings to better understand the impact of air-liquid interfacial phenomena on enzymatic hydrolysis of cellulose contained in Avicel, Sigmacell, α-cellulose, cotton linters, and filter paper. The impacts of solids and enzyme loadings, supplementation with nonionic surfactant Tween 20 and xylanases, and application of different types of mixing and reactor designs on cellulose hydrolysis were also evaluated.ResultsAvicel cellulose conversions at high solid loading were more than doubled by minimizing loss of cellulases to the air-liquid interface. Maximum cellulose conversions were high for surface-active supplemented shaken flasks or unshaken flasks because of low cellulase deactivation at the air-liquid interface. The nonionic surfactant Tween 20 was unable to completely prevent cellulase deactivation in shaken flasks and only reduced cellulose conversions at unreasonably high concentrations.ConclusionsHigh dynamic interfacial areas created through baffles in reactor vessels, low volumes in high-capacity vessels, or high shaking speeds severely limited cellulose conversions at low enzyme loadings. Precipitation of cellulases due to aggregation at the air-liquid interface caused their continuous deactivation in shaken flasks and severely limited solubilization of cellulose.

BackgroundPretreatment is effective in reducing the natural recalcitrance of plant biomass so polysaccharides in cell walls can be accessed for conversion to sugars. Furthermore, lignocellulosic biomass must typically be reduced in size to increase the pretreatment effectiveness and realize high sugar yields. However, biomass size reduction is a very energy-intensive operation and contributes significantly to the overall capital cost.ResultsIn this study, the effect of particle size reduction and biomass presoaking on the deconstruction of Alamo switchgrass was examined prior to pretreatment by dilute sulfuric acid (DSA) and Co-solvent Enhanced Lignocellulosic Fractionation (CELF) at pretreatment conditions optimized for maximum sugar release by each pretreatment coupled with subsequent enzymatic hydrolysis. Sugar yields by enzymatic hydrolysis were measured over a range of enzyme loadings. In general, DSA successfully solubilized hemicellulose, while CELF removed nearly 80% of Klason lignin from switchgrass in addition to the majority of hemicellulose. Presoaking and particle size reduction did not have a significant impact on biomass compositions after pretreatment for both DSA and CELF. However, presoaking for 4 h slightly increased sugar yields by enzymatic hydrolysis of DSA-pretreated switchgrass compared to unsoaked samples, whereas sugar yields from enzymatic hydrolysis of CELF solids continued to increase substantially for up to 18 h of presoaking time. Of particular importance, DSA required particle size reduction by knife milling to

[This corrects the article DOI: 10.1186/s13068-019-1353-7.].

[This corrects the article DOI: 10.1186/s13068-019-1353-7.].

Background:In this work, three pretreatments under investigation at the DOE Bioenergy Research Centers (BRCs) were subjected to a side-by-side comparison to assess their performance on model bioenergy hardwoods (a eucalyptus and a hybrid poplar). These include co-solvent-enhanced lignocellulosic fractionation (CELF), pretreatment with an ionic liquid using potentially biomass-derived components (cholinium lysinate or [Ch][Lys]), and two-stage Cu-catalyzed alkaline hydrogen peroxide pretreatment (Cu-AHP). For each of the feedstocks, the pretreatments were assessed for their impact on lignin and xylan solubilization and enzymatic hydrolysis yields as a function of enzyme loading. Lignins recovered from the pretreatments were characterized for polysaccharide content, molar mass distributions, β-aryl ether content, and response to depolymerization by thioacidolysis. Results:All three pretreatments resulted in significant solubilization of lignin and xylan, with the CELF pretreatment solubilizing the majority of both biopolymer categories. Enzymatic hydrolysis yields were shown to exhibit a strong, positive correlation with the lignin solubilized for the low enzyme loadings. The pretreatment-derived solubles in the [Ch][Lys]-pretreated biomass were presumed to contribute to inhibition of enzymatic hydrolysis in the eucalyptus as a substantial fraction of the pretreatment liquor was carried forward into hydrolysis for this pretreatment. The pretreatment-solubilized lignins exhibited significant differences in polysaccharide content, molar mass distributions, aromatic monomer yield by thioacidolysis, and β-aryl ether content. Key trends include a substantially higher polysaccharide content in the lignins recovered from the [Ch][Lys] pretreatment and high β-aryl ether contents and aromatic monomer yields from the Cu-AHP pretreatment. For all lignins, the 13C NMR-determined β-aryl ether content was shown to be correlated with the monomer yield with a second-order functionality. Conclusions:Overall, it was demonstrated that the three pretreatments highlighted in this study demonstrated uniquely different functionalities in reducing biomass recalcitrance and achieving higher enzymatic hydrolysis yields for the hybrid poplar while yielding a lignin-rich stream that may be suitable for valorization. Furthermore, modification of lignin during pretreatment, particularly cleavage of β-aryl ether bonds, is shown to be detrimental to subsequent depolymerization.

In the shadow of a burgeoning biomass-to-fuels industry, biological conversion of lignocellulose to fermentable sugars in a cost-effective manner is key to the success of second-generation and advanced biofuel production. For the effective comparison of one cellulase preparation to another, cellulase assays are typically carried out with one or more engineered cellulase formulations or natural exoproteomes of known performance serving as positive controls. When these formulations have unknown composition, as is the case with several widely used commercial products, it becomes impossible to compare or reproduce work done today to work done in the future, where, for example, such preparations may not be available. Therefore, being a critical tenet of science publishing, experimental reproducibility is endangered by the continued use of these undisclosed products. We propose the introduction of standard procedures and materials to produce specific and reproducible cellulase formulations. These formulations are to serve as yardsticks to measure improvements and performance of new cellulase formulations.

In the shadow of a burgeoning biomass-to-fuels industry, biological conversion of lignocellulose to fermentable sugars in a cost-effective manner is key to the success of second-generation and advanced biofuel production. For the effective comparison of one cellulase preparation to another, cellulase assays are typically carried out with one or more engineered cellulase formulations or natural exoproteomes of known performance serving as positive controls. When these formulations have unknown composition, as is the case with several widely used commercial products, it becomes impossible to compare or reproduce work done today to work done in the future, where, for example, such preparations may not be available. Therefore, being a critical tenet of science publishing, experimental reproducibility is endangered by the continued use of these undisclosed products. We propose the introduction of standard procedures and materials to produce specific and reproducible cellulase formulations. These formulations are to serve as yardsticks to measure improvements and performance of new cellulase formulations.

BackgroundIn a biorefinery producing cellulosic biofuels, biomass pretreatment will significantly influence the efficacy of enzymatic hydrolysis and microbial fermentation. Comparison of different biomass pretreatment techniques by studying the impact of pretreatment on downstream operations at industrially relevant conditions and performing comprehensive mass balances will help focus attention on necessary process improvements, and thereby help reduce the cost of biofuel production.ResultsAn on-going collaboration between the three US Department of Energy (DOE) funded bioenergy research centers (Great Lakes Bioenergy Research Center (GLBRC), Joint BioEnergy Institute (JBEI) and BioEnergy Science Center (BESC)) has given us a unique opportunity to compare the performance of three pretreatment processes, notably dilute acid (DA), ionic liquid (IL) and ammonia fiber expansion (AFEX(TM)), using the same source of corn stover. Separate hydrolysis and fermentation (SHF) was carried out using various combinations of commercially available enzymes and engineered yeast (Saccharomyces cerevisiae 424A) strain. The optimal commercial enzyme combination (Ctec2: Htec2: Multifect Pectinase, percentage total protein loading basis) was evaluated for each pretreatment with a microplate-based assay using milled pretreated solids at 0.2% glucan loading and 15 mg total protein loading/g of glucan. The best enzyme combinations were 67:33:0 for DA, 39:33:28 for IL and 67:17:17 for AFEX. The amounts of sugar (kg) (glucose: xylose: total gluco- and xylo-oligomers) per 100 kg of untreated corn stover produced after 72 hours of 6% glucan loading enzymatic hydrolysis were: DA (25:2:2), IL (31:15:2) and AFEX (26:13:7). Additionally, the amounts of ethanol (kg) produced per 100 kg of untreated corn stover and the respective ethanol metabolic yield (%) achieved with exogenous nutrient supplemented fermentations were: DA (14.0, 92.0%), IL (21.2, 93.0%) and AFEX (20.5, 95.0%), respectively. The reason for lower ethanol yield for DA is because most of the xylose produced during the pretreatment was removed and not converted to ethanol during fermentation.ConclusionsCompositional analysis of the pretreated biomass solids showed no significant change in composition for AFEX treated corn stover, while about 85% of hemicellulose was solubilized after DA pretreatment, and about 90% of lignin was removed after IL pretreatment. As expected, the optimal commercial enzyme combination was different for the solids prepared by different pretreatment technologies. Due to loss of nutrients during the pretreatment and washing steps, DA and IL pretreated hydrolysates required exogenous nutrient supplementation to ferment glucose and xylose efficiently, while AFEX pretreated hydrolysate did not require nutrient supplementation.