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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.

Cellulose reducing ends are believed to play a vital role in the cellulose recalcitrance to enzymatic conversion. However, their role in insoluble cellulose accessibility and hydrolysis is not clear. Thus, in this study, reducing ends of insoluble cellulose derived from various sources were modified by applying reducing and/or oxidizing agents. The effects of cellulose reducing ends modification on cellulose reducing ends, cellulose structure, and cellulose accessibility to cellulase were evaluated along with the impact on cellulose hydrolysis with complete as well purified cellulase components. Sodium borohydride (NaBH4) reduction and sodium chlorite‐acetic acid (SC/AA) oxidation were able to modify more than 90% and 60% of the reducing ends, respectively, while the bicinchoninic acid (BCA) reagent applied for various cycles oxidized cellulose reducing ends to various extents. X‐ray diffractograms of the treated solids showed that these treatments did not change the cellulose crystalline structure and the change in crystallinity index was insignificant. Surprisingly, it was found that the cellulose reducing ends modification, either through selective NaBH4 reduction or BCA oxidation, had a negligible impact on cellulose accessibility as well on cellulose hydrolysis rates or final conversions with complete cellulase at loadings as low as 0.5 mg protein/g cellulose. In fact, in contrast to what is traditionally believed, modifications of cellulose reducing ends by these two methods had no apparent impact on cellulose conversion with purified cellulase components and their synergy. However, SC/AA oxidation resulted in significant drop in cellulose conversion (10%–50%) with complete as well purified cellulase components. Nonetheless, further research revealed that the cause for drop in cellulose conversion for the SC/AA oxidation case was due to primary hydroxyl groups (PHGs) oxidation and not the oxidation of reducing ends. Furthermore, it was found that the PHGs modification affects cellulose accessibility and slows the cellulase uptake as well resulting in significant drop in cellulose conversions. [ABSTRACT FROM AUTHOR]

Simultaneous saccharification and fermentation (SSF) of solid biomass can reduce the complexity and improve the economics of lignocellulosic ethanol production by consolidating process steps and reducing end-product inhibition of enzymes compared with separate hydrolysis and fermentation (SHF). However, a longstanding limitation of SSF has been too low ethanol yields at the high-solids loading of biomass needed during fermentation to realize sufficiently high ethanol titers favorable for more economical ethanol recovery. Here, we illustrate how competing factors that limit ethanol yields during high-solids fermentations are overcome by integrating newly developed cosolvent-enhanced lignocellulosic fractionation (CELF) pretreatment with SSF. First, fed-batch glucose fermentations by Saccharomyces cerevisiae D5A revealed that this strain, which has been favored for SSF, can produce ethanol at titers of up to 86 g.L-1. Then, optimizing SSF of CELF-pretreated corn stover achieved unprecedented ethanol titers of 79.2, 81.3 and 85.6 g.L-1 in batch shake flask, corresponding to ethanol yields of 90.5%, 86.1% and 80.8% at solids loadings of 20.0 wt %, 21.5 wt % and 23.0 wt %, respectively. Ethanol yields remained at over 90% despite reducing enzyme loading to only 10 mg protein.g glucan-1 [∼6.5 filter paper units (FPU)], revealing that the enduring factors limiting further ethanol production were reduced cell viability and glucose uptake by D5A and not loss of enzyme activity or mixing issues, thereby demonstrating an SSF-based process that was limited by a strain's metabolic capabilities and tolerance to ethanol. [ABSTRACT FROM AUTHOR]

Background: Woody biomass is highly recalcitrant to enzymatic sugar release and often requires significant size reduction and severe pretreatments to achieve economically viable sugar yields in biological production of sustainable fuels and chemicals. However, because mechanical size reduction of woody biomass can consume significant amounts of energy, it is desirable to minimize size reduction and instead pretreat larger wood chips prior to biological conversion. To date, however, most laboratory research has been performed on materials that are significantly smaller than applicable in a commercial setting. As a result, there is a limited understanding of the effects that larger biomass particle size has on the effectiveness of steam explosion pretreatment and subsequent enzymatic hydrolysis of wood chips. Results: To address these concerns, novel downscaled analysis and high throughput pretreatment and hydrolysis (HTPH) were applied to examine whether differences exist in the composition and digestibility within a single pretreated wood chip due to heterogeneous pretreatment across its thickness. Heat transfer modeling, Simons' stain testing, magnetic resonance imaging (MRI), and scanning electron microscopy (SEM) were applied to probe the effects of pretreatment within and between pretreated wood samples to shed light on potential causes of variation, pointing to enzyme accessibility (i.e., pore size) distribution being a key factor dictating enzyme digestibility in these samples. Application of these techniques demonstrated that the effectiveness of pretreatment of Populus tremuloides can vary substantially over the chip thickness at short pretreatment times, resulting in spatial digestibility effects and overall lower sugar yields in subsequent enzymatic hydrolysis. Conclusions: These results indicate that rapid decompression pretreatments (e.g., steam explosion) that specifically alter accessibility at lower temperature conditions are well suited for larger wood chips due to the non-uniformity in temperature and digestibility profiles that can result from high temperature and short pretreatment times. Furthermore, this study also demonstrated that wood chips were hydrated primarily through the natural pore structure during pretreatment, suggesting that preserving the natural grain and transport systems in wood during storage and chipping processes could likely promote pretreatment efficacy and uniformity. [ABSTRACT FROM AUTHOR]

We introduce a new pretreatment called co-solvent-enhanced lignocellulosic fractionation (CELF) to reduce enzyme costs dramatically for high sugar yields from hemicellulose and cellulose, which is essential for the low-cost conversion of biomass to fuels. CELF employs THF miscible with aqueous dilute acid to obtain up to 95 % theoretical yield of glucose, xylose, and arabinose from corn stover even if coupled with enzymatic hydrolysis at only 2 mgenzyme gglucan−1. The unusually high saccharification with such low enzyme loadings can be attributed to a very high lignin removal, which is supported by compositional analysis, fractal kinetic modeling, and SEM imaging. Subsequently, nearly pure lignin product can be precipitated by the evaporation of volatile THF for recovery and recycling. Simultaneous saccharification and fermentation of CELF-pretreated solids with low enzyme loadings and Saccharomyces cerevisiae produced twice as much ethanol as that from dilute-acid-pretreated solids if both were optimized for corn stover. [ABSTRACT FROM AUTHOR]

To maximize yields of fermentable xylose monomer from hydrothermal pretreatment of corn stover at 200 °C, the xylooligomers-rich liquor produced was post hydrolyzed with dilute sulfuric acid over a range of times and acid concentrations. The results showed that application of 0.75% H2SO4 at 110 °C for 180 min or 0.50% H2SO4 at 110 °C for 240 min recovered almost 100% of the total xylose from the oligomers. Furthermore, adjusting one constant in the combined severity parameter (CSP) provided a rapid and accurate tool for trading off times, temperatures, and acid concentrations to reach the highest xylose yields from xylooligomers in hydrothermal pretreatment solutions. This adjusted CSP showed that an 8.4 °C temperature increase has the same impact as doubling the acid concentration or halving reaction time instead of the 10 °C change projected by the customary CSP and suggests that xylooligomers bonds are more easily broken than the hemicellulose from which they are derived. [ABSTRACT FROM AUTHOR]

Background Pretreatment is essential to realize high product yields from biological conversion of naturally recalcitrant cellulosic biomass, with thermochemical pretreatments often favored for cost and performance. In this study, enzymatic digestion of solids from dilute sulfuric acid (DA), ammonia fiber expansion (AFEXTM), and ionic liquid (IL) thermochemical pretreatments of corn stover were followed over time for the same range of total enzyme protein loadings to provide comparative data on glucose and xylose yields of monomers and oligomers from the pretreated solids. The composition of pretreated solids and enzyme adsorption on each substrate were also measured to determine. The extent glucose release could be related to these features. Results Corn stover solids from pretreatment by DA, AFEX, and IL were enzymatically digested over a range of low to moderate loadings of commercial cellulase, xylanase, and pectinase enzyme mixtures, the proportions of which had been previously optimized for each pretreatment. Avicel® cellulose, regenerated amorphous cellulose (RAC), and beechwood xylan were also subjected to enzymatic hydrolysis as controls. Yields of glucose and xylose and their oligomers were followed for times up to 120 hours, and enzyme adsorption was measured. IL pretreated corn stover displayed the highest initial glucose yields at all enzyme loadings and the highest final yield for a low enzyme loading of 3 mg protein/g glucan in the raw material. However, increasing the enzyme loading to 12 mg/g glucan or more resulted in DA pretreated corn stover attaining the highest longer-term glucose yields. Hydrolyzate from AFEX pretreated corn stover had the highest proportion of xylooligomers, while IL produced the most glucooligomers. However, the amounts of both oligomers dropped with increasing enzyme loadings and hydrolysis times. IL pretreated corn stover had the highest enzyme adsorption capacity. Conclusions Initial hydrolysis yields were highest for substrates with greater lignin removal, a greater degree of change in cellulose crystallinity, and high enzyme accessibility. Final glucose yields could not be clearly related to concentrations of xylooligomers released from xylan during hydrolysis. Overall, none of these factors could completely account for differences in enzymatic digestion performance of solids produced by AFEX, DA, and IL pretreatments. [ABSTRACT FROM AUTHOR]

Background Agave, which is well known for tequila and other liquor production in Mexico, has recently gained attention because of its attractive potential to launch sustainable bioenergy feedstock solutions for semi-arid and arid lands. It was previously found that agave cell walls contain low lignin and relatively diverse non-cellulosic polysaccharides, suggesting unique recalcitrant features when compared to conventional C4 and C3 plants. Results Here, we report sugar release data from fungal enzymatic hydrolysis of non-pretreated and hydrothermally pretreated biomass that shows agave to be much less recalcitrant to deconstruction than poplar or switchgrass. In fact, non-pretreated agave has a sugar release five to eight times greater than that of poplar wood and switchgrass . Meanwhile, state of the art techniques including glycome profiling, nuclear magnetic resonance (NMR), Simon's Stain, confocal laser scanning microscopy and so forth, were applied to measure interactions of non-cellulosic wall components, cell wall hydrophilicity, and enzyme accessibility to identify key structural features that make agave cell walls less resistant to biological deconstruction when compared to poplar and switchgrass. Conclusions This study systematically evaluated the recalcitrant features of agave plants towards biofuels applications. The results show that not only does agave present great promise for feeding biorefineries on semi-arid and arid lands, but also show the value of studying agave's low recalcitrance for developments in improving cellulosic energy crops. [ABSTRACT FROM AUTHOR]

A novel single phase co-solvent system using tetrahydrofuran (THF) promotes hydrolysis of maple wood to sugars, sugar dehydration, and lignin extraction simultaneously and achieves higher overall yields of the fuel precursors furfural, 5-hydroxymethylfurfural (HMF), and levulinic acid (LA) than previously reported from biomass. In a one-pot reaction, we obtained yields of 86% furfural, 21% HMF, and 40% LA in the liquid phase and over 90% extraction of lignin as a solid powder. The co-solvent reaction also produced a glucan-rich residue that is highly digestible by enzymes for biological conversion to ethanol or further thermochemical reaction to additional HMF and levulinic acid. These findings enable an integrated conversion platform in which THF is both a co-solvent and final co-product to enhance production of fuel precursors for catalytic upgrading to renewable liquid hydrocarbons fuels. [ABSTRACT FROM AUTHOR]

Dilute acid as well as water only (hydrothermal) pretreatments often lead to a significant hemicellulose loss to soluble furans and insoluble degradation products, collectively termed as chars and/or pseudo-lignin. In order to understand the factors contributing to reducing sugar yields from pretreated biomass and the possible influence of hemicellulose derived pseudo-lignin on cellulose conversion at the moderate to low enzyme loadings necessary for favorable economics, dilute acid pretreatment of Avicel cellulose alone and mixed with beechwood xylan or xylose was performed at various severities. Following pretreatment, the solids were enzymatically hydrolyzed and characterized for chemical composition and physical properties by NMR, FT-IR, and SEM imaging. It was found that hemicelluloses (xylan) derived-pseudo-lignin was formed at even moderate severities and that these insoluble degradation products can significantly retard cellulose hydrolysis. Furthermore, although low severity (CSF ∼ 1.94) dilute acid pretreatment of a xylan-Avicel mixture hydrolyzed most of the xylan (98%) and produced negligible amounts of pseudo-lignin, enzymatic conversion of cellulose dropped significantly (>25%) compared to cellulose pretreated alone at the same conditions. The drop in cellulose conversion was higher than realized for cellulase inhibition by xylooligomers reported previously. Plausible mechanisms are discussed to explain the observed reductions in cellulose conversions. Biotechnol. Bioeng. 2013; 110: 737-753. © 2012 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Background: Because many Miscanthus genotypes can be cultivated with relatively high productivity and carbohydrate content, Miscanthus has great potential as an energy crop that can support large scale biological production of biofuels. Results: In this study, batch hydrothermal pretreatment at 180°C for 35 min followed by enzymatic hydrolysis was shown to give the highest total sugar yields for Miscanthus x giganteus cv. Illinois planted in Illinois. High throughput pretreatment at 180°C for 35 min and 17.5 min followed by co-hydrolysis in a multi-well batch reactor identified two varieties out of 80 that had significantly higher sugar yields from pretreatment and enzymatic hydrolysis than others. The differences in performance were then related to compositions of the 80 varieties to provide insights into desirable traits for Miscanthus that enhance sugar yields. Conclusions: High throughput pretreatment and co-hydrolysis (HTPH) rapidly identified promising genotypes from a wide range of Miscanthus genotypes, including hybrids of Miscanthus sacchariflorus/M. sinensis and Miscanthus lutarioriparius, differentiating the more commercially promising species from the rest. The total glucan plus xylan content in Miscanthus appeared to influence both mass and theoretical yields, while lignin and ash contents did not have a predictable influence on performance. [ABSTRACT FROM AUTHOR]

In this study, the kinetics of the acid catalyzed hydrolysis of microcrystalline cellulose (Avicel PH101) to levulinic (LA) and formic (FA) acids was investigated in a batch reactor over the following range of conditions: 160-200°C, hydrochloric acid concentrations of 0.309-0.927 M (11.3-33.8 g/l), cellulose concentrations of 49.8-149 mM (8.06-24.1 g/l), and residence times of 0-50 min. The maximum LA yield of around 60% of theoretical was achieved for an initial cellulose concentration of 99.6 mM, acid concentration 0.927 M, and 180-200°C. A mathematical model and its analytical solution were developed to predict conversion of cellulose to LA and FA through glucose and hydroxymethyl-2-furfural based on an irreversible pseudo-first order reaction. Rate analysis of each reaction indicated that the rate-controlling step shifted from LA formation initially to HMF formation later. © 2011 American Institute of Chemical Engineers AIChE J, 2012 [ABSTRACT FROM AUTHOR]

Abstract: Dilute sulfuric acid (DA), sulfur dioxide (SO2), liquid hot water (LHW), soaking in aqueous ammonia (SAA), ammonia fiber expansion (AFEX), and lime pretreatments were applied to Alamo, Dacotah, and Shawnee switchgrass. Application of the same analytical methods and material balance approaches facilitated meaningful comparisons of glucose and xylose yields from combined pretreatment and enzymatic hydrolysis. Use of a common supply of cellulase, beta-glucosidase, and xylanase also eased comparisons. All pretreatments enhanced sugar recovery from pretreatment and subsequent enzymatic hydrolysis substantially compared to untreated switchgrass. Adding beta-glucosidase was effective early in enzymatic hydrolysis while cellobiose levels were high but had limited effect on longer term yields at the enzyme loadings applied. Adding xylanase improved yields most for higher pH pretreatments where more xylan was left in the solids. Harvest time had more impact on performance than switchgrass variety, and microscopy showed changes in different features could impact performance by different pretreatments. [Copyright &y& Elsevier]

The primary obstacle to producing renewable fuels from lignocellulosic biomass is a plant's recalcitrance to releasing sugars bound in the cell wall. From a sample set of wood cores representing 1,100 individual undomesticated Populus trichocarpa trees, 47 extreme phenotypes were selected across measured lignin content and ratio of syringyl and guaiacyl units (S/G ratio). This subset was tested for total sugar release through enzymatic hydrolysis alone as well as through combined hot-water pretreatment and enzymatic hydrolysis using a high-throughput screening method. The total amount of glucan and xylan released varied widely among samples, with total sugar yields of up to 92% of the theoretical maximum. A strong negative correlation between sugar release and lignin content was only found for pretreated samples with an S/G ratio < 2.0. For higher S/G ratios, sugar release was generally higher, and the negative influence of lignin was less pronounced. When examined separately, only glucose release was correlated with lignin content and S/G ratio in this manner, whereas xylose release depended on the S/G ratio alone. For enzymatic hydrolysis without pretreatment, sugar release increased significantly with decreasing lignin content below 20%, irrespective of the S/G ratio. Furthermore, certain samples featuring average lignin content and S/G ratios exhibited exceptional sugar release. These facts suggest that factors beyond lignin and S/G ratio influence recalcitrance to sugar release and point to a critical need for deeper understanding of cell-wall structure before plants can be rationally engineered for reduced recalcitrance and efficient biofuels production. [ABSTRACT FROM AUTHOR]

Abstract: 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. [ABSTRACT FROM AUTHOR]

Abstract: Understanding structural characteristics that are responsible for biomass recalcitrance by identifying why it is more difficult for some plants, or portions of plants, to release their sugars would be extremely valuable in overcoming this barrier. With this in mind, this study investigated the recalcitrance of wood by considering the effects of aging in two Populus tremuloides cross sections. By applying our novel small scale systems, including a multi-well pretreatment and enzymatic hydrolysis system and a downscaled compositional analysis procedure, we were able to follow ring-by-ring compositions and sugar release patterns. Observed variations were then related to structural changes that occur across the radial direction of trees, providing an important step toward understanding the influence of these changes on recalcitrance. [ABSTRACT FROM AUTHOR]

In this paper we demonstrate an integrated process for the production of high octane gasoline from maple wood by hydrolysis of maple wood into aqueous carbohydrate solutions followed by aqueous phase hydrodeoxygenation of the sugar solutions. The aqueous carbohydrate solutions were prepared by both hydrolysis in hot water and hydrolysis with dilute acids (H2SO4, oxalic acid). The aqueous carbohydrate solutions were a mixture of xylose, water soluble hemicellulose oligomers, acetic acid, glucose, glucose oligomers, and probably some lignin polymers. Hydrolysis with hot water produced primarily hemicellulose oligomers whereas hydrolysis with acids produced mainly xylose and acetic acid. The hydrolysis co-product was a solid enriched in cellulose and lignin. The aqueous streams were hydrodeoxygenated by a two step catalytic process in which the first catalyst bed contained a Ru/C catalyst at 393 K and the second catalyst bed contained a Pt/zirconium phosphate (Pt/ZrP) catalyst at 518 K. The Ru/C catalyst was able to selectively hydrogenate xylose into xylitol but could not selectively hydrogenate the xylose oligomers. The two stage process was able to convert the aqueous carbohydrate streams derived from maple wood into gasoline range products with carbon yields of up to 57% and an estimated octane number of 96.5. No significant catalyst deactivation was observed indicating that the catalysts are very stable. The highest gasoline yield from this two stage process was obtained from the stream produced by acid hydrolysis of maple wood with 0.5 wt% oxalic acid at 433 K for 0.5 h. These results suggest that aqueous phase processing of sugars obtained by hydrolysis is a promising option for the production of fuels and chemicals from lignocellulosic biomass. [ABSTRACT FROM AUTHOR]

Background: Hemicellulose 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. Results: In 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. Conclusions: This 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. [ABSTRACT FROM AUTHOR]

Abstract: Ethanol made biologically from a variety of cellulosic biomass sources such as agricultural and forestry residues, grasses, and fast growing wood is widely recognized as a unique sustainable liquid transportation fuel with powerful economic, environmental, and strategic attributes, but production costs must be competitive for these benefits to be realized. Continuous hydrolysis and fermentation processes offer important potential advantages in reducing costs, but little has been done on continuous processing of cellulosic biomass to ethanol. As shown in this review, some continuous fermentations are now employed for commercial ethanol production from cane sugar and corn to take advantage of higher volumetric productivity, reduced labor costs, and reduced vessel down time for cleaning and filling. On the other hand, these systems are more susceptible to microbial contamination and require more sophisticated operations. Despite the latter challenges, continuous processes could be even more important to reducing the costs of overcoming the recalcitrance of cellulosic biomass, the primary obstacle to low cost fuels, through improving the effectiveness of utilizing expensive enzymes. In addition, continuous processing could be very beneficial in adapting fermentative organisms to the wide range of inhibitors generated during biomass pretreatment or its acid catalyzed hydrolysis. If sugar generation rates can be increased, the high cell densities in a continuous system could enable higher productivities and yields than in batch fermentations. [Copyright &y& Elsevier]

The accessibility of cellulase and xylanase enzymes to glucan and xylan, respectively, and its change with conversion were measured for pure Avicel glucan and poplar solids that had been pretreated by ammonia fiber expansion (AFEX), ammonia recycled percolation (ARP), dilute acid, and lime. Avicel and pretreated solids were digested to various degrees by cellulase together with ¿-glucosidase enzymes and then cleaned of residual protein via a biological method using Protease. Glucan accessibility was determined by purified CBHI (Cel7A) adsorption at 4°C, and 4 and 24h hydrolysis yields were determined for solids loading containing equal amounts of glucan (1.0% w/v) and lignin (1.0% w/v), in two separate sets of experiments. Consistent with our previous study and in contrast to some in the literature, little change in glucan accessibility was observed with conversion for Avicel, but glucan and xylan accessibility for real biomass varied with the type of pretreatment. For example, AFEX pretreated solids showed a negligible change in glucan accessibility for conversion up to 90%, although xylan accessibility seemed to decline first and then remained constant. On the other hand, a substantial decline in glucan and xylan accessibility with conversion was observed for lime pretreated poplar solids, as shown by initial hydrolysis rates. Yet, an increase in CBHI adsorption with conversion for lime pretreated poplar solids suggested the opposite trend, possibly due to increased lignin exposure and/or reduced effectiveness of adsorbed enzyme. [Copyright &y& Elsevier]

The Biomass Refining Consortium for Applied Fundamentals and Innovation, with members from Auburn University, Dartmouth College, Michigan State University, the National Renewable Energy Laboratory, Purdue University, Texas A&M University, the University of British Columbia, and the University of California at Riverside, has developed comparative data on the conversion of corn stover to sugars by several leading pretreatment technologies. These technologies include ammonia fiber expansion pretreatment, ammonia recycle percolation pretreatment, dilute sulfuric acid pretreatment, flowthrough pretreatment (hot water or dilute acid), lime pretreatment, controlled pH hot water pretreatment, and sulfur dioxide steam explosion pretreatment. Over the course of two separate USDA- and DOE-funded projects, these pretreatment technologies were applied to two different corn stover batches, followed by enzymatic hydrolysis of the remaining solids from each pretreatment technology using identical enzyme preparations, enzyme loadings, and enzymatic hydrolysis assays. Identical analytical methods and a consistent material balance methodology were employed to develop comparative sugar yield data for each pretreatment and subsequent enzymatic hydrolysis. Although there were differences in the profiles of sugar release, with the more acidic pretreatments releasing more xylose directly in the pretreatment step than the alkaline pretreatments, the overall glucose and xylose yields (monomers + oligomers) from combined pretreatment and enzymatic hydrolysis process steps were very similar for all of these leading pretreatment technologies. Some of the water-only and alkaline pretreatment technologies resulted in significant amounts of residual xylose oligomers still remaining after enzymatic hydrolysis that may require specialized enzyme preparations to fully convert xylose oligomers to monomers. [ABSTRACT FROM AUTHOR]

Comparative data is presented on glucose and xylose release for enzymatic hydrolysis of solids produced by pretreatment of poplar wood by ammonia fiber expansion (AFEX), ammonia recycled percolation (ARP), controlled pH, dilute acid, flowthrough (FT), lime, and sulfur dioxide (SO2) technologies. Sugar solubilization was measured for times of up to 72 h using cellulase supplemented with β-glucosidase at an activity ratio of 1:2, respectively, at combined protein mass loadings of 5.8-116 mg/g of glucan in poplar wood prior to pretreatment. In addition, the enzyme cocktail was augmented with up to 11.0 g of xylanase protein per gram of cellulase protein at combined cellulase and β-glucosidase mass loadings of 14.5 and 29.0 mg protein (about 7.5 and 15 FPU, respectively)/g of original potential glucose to evaluate cellulase-xylanase interactions. All pretreated poplar solids required high protein loadings to realize good sugar yields via enzymatic hydrolysis, and performance tended to be better for low pH pretreatments by dilute sulfuric acid and sulfur dioxide, possibly due to higher xylose removal. Glucose release increased nearly linearly with residual xylose removal by enzymes for all pretreatments, xylanase leverage on glucan removal decreased at high cellulase loadings. Washing the solids improved digestion for all pretreatments and was particularly beneficial for controlled pH pretreatment. Furthermore, incubation of pretreated solids with BSA, Tween 20, or PEG6000 prior to adding enzymes enhanced yields, but the effectiveness of these additives varied with the type of pretreatment. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [ABSTRACT FROM AUTHOR]

Through a Biomass Refining Consortium for Applied Fundamentals and Innovation among Auburn University, Dartmouth College, Michigan State University, the National Renewable Energy Laboratory, Purdue University, Texas A&M University, the University of British Columbia, and the University of California at Riverside, leading pretreatment technologies based on ammonia fiber expansion, aqueous ammonia recycle, dilute sulfuric acid, lime, neutral pH, and sulfur dioxide were applied to a single source of poplar wood, and the remaining solids from each technology were hydrolyzed to sugars using the same enzymes. Identical analytical methods and a consistent material balance methodology were employed to develop comparative performance data for each combination of pretreatment and enzymes. Overall, compared to data with corn stover employed previously, the results showed that poplar was more recalcitrant to conversion to sugars and that sugar yields from the combined operations of pretreatment and enzymatic hydrolysis varied more among pretreatments. However, application of more severe pretreatment conditions gave good yields from sulfur dioxide and lime, and a recombinant yeast strain fermented the mixed stream of glucose and xylose sugars released by enzymatic hydrolysis of water washed solids from all pretreatments to ethanol with similarly high yields. An Agricultural and Industrial Advisory Board followed progress and helped steer the research to meet scientific and commercial needs. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [ABSTRACT FROM AUTHOR]

Mechanisms that control xylan removal during pretreatment of lignocellulosic biomass are not well understood. For example, although hemicellulose hydrolysis is virtually always assumed to follow first-order homogeneous kinetics, the increase in xylan removal with flow rate for flowthrough pretreatment systems is inconsistent with the predictions for such models, and better information is needed to understand the causes of such discrepancies. Thus, new methods were developed to follow the fate of xylooligomers with degrees of polymerization of up to 30, a range not possible before, for water-only flowthrough pretreatment of oat spelt xylan and corn stover for temperatures of 200–240°C. Material balances based on the oligomer release profiles produced by batch and flowthrough operations could be closed, suggesting the methods were quite accurate. However, the results also showed that increasing the flow rate from 0 to 2 and then 25mL/min affected the size distribution of the xylan oligomers (DP<30) released from corn stover but not from oat spelt xylan and also increased overall hemicellulose sugar solubilization. One explanation for these difference is that lignin and lignin–xylan compounds in particular play an important role in the hydrolysis of lignocellulosic biomass. [Copyright &y& Elsevier]

Abstract: Biological processing of cellulosic biomass to fuels and chemicals would open up major new agricultural markets and provide powerful societal benefits, but pretreatment operations essential to economically viable yields have a major impact on costs and performance of the entire system. However, little comparative data is available on promising pretreatments. To aid in selecting appropriate systems, leading pretreatments based on ammonia explosion, aqueous ammonia recycle, controlled pH, dilute acid, flowthrough, and lime were evaluated in a coordinated laboratory program using a single source of corn stover, the same cellulase enzyme, shared analytical methods, and common data interpretation approaches to make meaningful comparisons possible for the first time. Each pretreatment made it possible to subsequently achieve high yields of glucose from cellulose by cellulase enzymes, and the cellulase formulations used were effective in solubilizing residual xylan left in the solids after each pretreatment. Thus, overall sugar yields from hemicellulose and cellulose in the coupled pretreatment and enzymatic hydrolysis operations were high for all of the pretreatments with corn stover. In addition, high-pH methods were found to offer promise in reducing cellulase use provided hemicellulase activity can be enhanced. However, the substantial differences in sugar release patterns in the pretreatment and enzymatic hydrolysis operations have important implications for the choice of process, enzymes, and fermentative organisms. [Copyright &y& Elsevier]

Abstract: Flowthrough pretreatment with compressed-hot water can significantly increase the removal of xylan and lignin and enhance xylose sugar yields and cellulose digestibility, especially at high flow rates. However, continuous flowthrough operations that realize these benefits suffer from a large amount of water consumption that leads to high energy requirements for pretreatment and downstream processing. Because high flow rates are particularly effective early in hemicellulose hydrolysis and less effective later, flow with compressed-hot water was applied at selected intervals, and performance was compared with that of batch and flowthrough operations for corn stover pretreated with compressed-hot water at 200°C. Partial flow reduced water consumption by 60% compared with continuous flowthrough operation but still achieved higher xylose sugar yields (84–89%) compared to batch pretreatment (46.6%). In addition, corn stover cellulose pretreated by partial flow had higher enzymatic digestibility (88–90%) than batch operations (∼85%) at otherwise identical conditions, apparently due to much higher lignin removal for the former (40–45% vs 10–12%). Partial flow also reduced degradation, with recovery of xylose and glucose in the solids and hydrolyzate increased to 90–92% vs only about 76% for batch operation. The partial flow approach could be further improved by optimizing the operating strategy and reaction conditions, suggesting that this novel pretreatment could lead to advanced biomass pretreatment technology. [Copyright &y& Elsevier]

Abstract: For the first time, a single source of cellulosic biomass was pretreated by leading technologies using identical analytical methods to provide comparative performance data. In particular, ammonia explosion, aqueous ammonia recycle, controlled pH, dilute acid, flowthrough, and lime approaches were applied to prepare corn stover for subsequent biological conversion to sugars through a Biomass Refining Consortium for Applied Fundamentals and Innovation (CAFI) among Auburn University, Dartmouth College, Michigan State University, the National Renewable Energy Laboratory, Purdue University, and Texas A&M University. An Agricultural and Industrial Advisory Board provided guidance to the project. Pretreatment conditions were selected based on the extensive experience of the team with each of the technologies, and the resulting fluid and solid streams were characterized using standard methods. The data were used to close material balances, and energy balances were estimated for all processes. The digestibilities of the solids by a controlled supply of cellulase enzyme and the fermentability of the liquids were also assessed and used to guide selection of optimum pretreatment conditions. Economic assessments were applied based on the performance data to estimate each pretreatment cost on a consistent basis. Through this approach, comparative data were developed on sugar recovery from hemicellulose and cellulose by the combined pretreatment and enzymatic hydrolysis operations when applied to corn stover. This paper introduces the project and summarizes the shared methods for papers reporting results of this research in this special edition of Bioresource Technology. [Copyright &y& Elsevier]

Ethanol made from lignocellulosic biomass sources, such as agricultural and forestry residues and herbaceous and woody crops, provides unique environmental, economic, and strategic benefits. Through sustained research funding, primarily by the U.S. Department of Energy, the estimated cost of biomass ethanol production has dropped from ∼$4.63/gallon in 1980 to ∼$1.22/gallon today, and it is now potentially competitive for blending with gasoline. Advances in pretreatment by acid-catalyzed hemicellulose hydrolysis and enzymes for cellulose breakdown coupled with recent development of genetically engineered bacteria that ferment all five sugars in biomass to ethanol at high yields have been the key to reducing costs. However, through continued advances in accessing the cellulose and hemicellulose fractions, the cost of biomass ethanol can be reduced to the point at which it is competitive as a pure fuel without subsidies. A major challenge to realizing the great benefits of biomass ethanol remains to substantially reduce the risk of commercializing first-of-a-kind technology, and greater emphasis on developing a fundamental understanding of the technology for biomass conversion to ethanol would reduce application costs and accelerate commercialization. Teaming of experts to cooperatively research key processing steps would be a particularly powerful and effective approach to meeting these needs. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The Inside Cover depicts a novel cellulosic biomass pretreatmENt called co ‐ solvENt ‐ ENhanced lignocellulosic fractionation (CELF) that uses THF to greatly ENhance sugar and ethanol yields from dilute acid pretreatmENt of corn stover. CELF removed and recovered hemicellulose sugars and most of the lignin to produce highly digestible cellulose that was hydrolyzed to glucose using 90   % less ENzymes than dilute ‐ acid pretreatmENt. Sugar yields from hemicellulose and cellulose were over 95   % of theoretical, and over 90   % ethanol yields were obtained by simultaneous saccharification and fermENtation (SSF). More details can be found in the Full Paper by NguyEN et al. (DOI: 10.1002/cssc.201403045). [ABSTRACT FROM AUTHOR]

The NREL standard procedure for lignocellulosic biomass composition has two steps: primary hydrolysis in 72% wt sulfuric acid at 30 °C for 1 h followed by secondary hydrolysis of the slurry in 4 wt% acid at 121 °C for 1 h. Although pointed out in the NREL procedure, the impact of particle size on composition has never been shown. In addition, the effects of primary hydrolysis time and separation of solids prior to secondary hydrolysis on composition have never been shown. Using poplar, it was found that particle sizes less than 0.250 mm significantly lowered the glucan content and increased the Klason lignin but did not affect xylan, acetate, or acid soluble lignin contents. Composition was unaffected for primary hydrolysis time between 30 and 90 min. Moreover, separating solids prior to secondary hydrolysis had negligible effect on composition suggesting that lignin and polysaccharides are completely separated in the primary hydrolysis stage. [ABSTRACT FROM AUTHOR]

Abstract: Dilute oxalic acid pretreatment was applied to maple wood to improve compatibility with downstream operations, and its performance in pretreatment and subsequent enzymatic hydrolysis was compared to results for hydrothermal and dilute hydrochloric and sulfuric acid pretreatments. The highest total xylose yield of ∼84% of the theoretical maximum was for both 0.5% oxalic and sulfuric acid pretreatment at 160°C, compared to ∼81% yield for hydrothermal pretreatment at 200°C and for 0.5% hydrochloric acid pretreatment at 140°C. The xylooligomer fraction from dilute oxalic acid pretreatment was only 6.3% of the total xylose in solution, similar to results with dilute hydrochloric and sulfuric acids but much lower than the ∼70% value for hydrothermal pretreatment. Combining any of the four pretreatments with enzymatic hydrolysis with 60FPUcellulase/g of glucan plus xylan in the pretreated maple wood resulted in virtually the same total glucose plus xylose yields of ∼85% of the maximum possible. [Copyright &y& Elsevier]

Abstract: Dacotah switchgrass was pretreated with sulfuric acid concentrations of 0.5, 1.0, and 2.0wt.% at 140, 160, and 180°C and with 1 and 3wt.% sulfur dioxide at 180°C over a range of times. Sulfur dioxide loadings of 0%, 1%, 3%, 5%, and 10%wt.% of dry biomass were also tested at 180°C for 10min. Sugar yields were tracked for pretreatment and subsequent enzymatic hydrolysis to identify conditions for the highest total sugar yields. Pretreatment with 1wt.% dilute sulfuric acid at 140°C for 40min followed by enzymatic hydrolysis with 48.6mg enzyme/g initial glucan in raw biomass resulted in ∼86% of theoretical yield for glucose and xylose combined. For sulfur dioxide pretreatment, the highest total sugar yield of about 87% occurred at 5% SO2 for 10min and 180°C. However, xylose yields were higher at shorter times and glucose yields at longer times. [Copyright &y& Elsevier]

Abstract: 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 fors 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 1.67mg/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 β-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. [ABSTRACT FROM AUTHOR]

Abstract: 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°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. [Copyright &y& Elsevier]

Previous studies defined easy and difficult to hydrolyze fractions of hemicellulose that may result from bonds among cellulose, hemicellulose, and lignin. To understand how such bonds affect hydrolysis, Populus trichocarpa × Populus deltoides , holocellulose isolated from P. trichocarpa × P. deltoides and birchwood xylan were subjected to hydrothermal flow-through pretreatment. Samples were characterized by glycome profiling, HPLC, and UPLC–MS. Glycome profiling revealed steady fragmentation and removal of glycans from solids during hydrolysis. The extent of polysaccharide fragmentation, hydrolysis rate, and total xylose yield were lowest for P. trichocarpa × P. deltoides and greatest for birchwood xylan. Comparison of results from P. trichocarpa × P. deltoides and holocellulose suggested that lignin–carbohydrate complexes reduce hydrolysis rates and limit release of large xylooligomers. Smaller differences between results with holocellulose and birchwood xylan suggest xylan–cellulose hydrogen bonds limited hydrolysis, but to a lesser extent. These findings imply cell wall structure strongly influences hydrolysis. [ABSTRACT FROM AUTHOR]

Abstract: We present a unique evaluation of three advanced high throughput pretreatment and enzymatic hydrolysis systems (HTPH-systems) for screening of lignocellulosic biomass for enzymatic saccharification. Straw from 20 cultivars of winter wheat from two sites in Denmark was hydrothermally pretreated and enzymatically processed in each of the separately engineered HTPH-systems at 1) University of California, Riverside, 2) National Renewable Energy Laboratory (NREL), Colorado, and 3) University of Copenhagen (CPH). All three systems were able to detect significant differences between the cultivars in the release of fermentable sugars, with average cellulose conversions of 57%, 64%, and 71% from Riverside, NREL and CPH, respectively. The best correlation of glucose yields was found between the Riverside and NREL systems (R 2 = 0.2139), and the best correlation for xylose yields was found between Riverside and CPH (R 2 = 0.4269). All three systems identified Flair as the highest yielding cultivar and Dinosor, Glasgow, and Robigus as low yielding cultivars. Despite different conditions in the three HTPH-systems, the approach of microscale screening for phenotypically less recalcitrant feedstock seems sufficiently robust to be used as a generic analytical platform. [Copyright &y& Elsevier]

Abstract: For this project, six chemical pretreatments were compared for the Consortium for Applied Fundamentals and Innovation (CAFI): ammonia fiber expansion (AFEX), dilute sulfuric acid (DA), lime, liquid hot water (LHW), soaking in aqueous ammonia (SAA), and sulfur dioxide (SO2). For each pretreatment, a material balance was analyzed around the pretreatment, optional post-washing step, and enzymatic hydrolysis of Dacotah switchgrass. All pretreatments+enzymatic hydrolysis solubilized over two-thirds of the available glucan and xylan. Lime, post-washed LHW, and SO2 achieved >83% total glucose yields. Lime, post-washed AFEX, and DA achieved >83% total xylose yields. Alkaline pretreatments, except AFEX, solubilized the most lignin and a portion of the xylan as xylo-oligomers. As pretreatment pH decreased, total solubilized xylan and released monomeric xylose increased. Low temperature-long time or high temperature-short time pretreatments are necessary for high glucose release from late-harvest Dacotah switchgrass but high temperatures may cause xylose degradation. [Copyright &y& Elsevier]

Abstract: The objective of this work is to investigate the effects of cellulase loading and β-glucosidase supplementation on enzymatic hydrolysis of pretreated Dacotah switchgrass. To assess the difference among various pretreatment methods, the profiles of sugars and intermediates were determined for differently treated substrates. For all pretreatments, 72h glucan/xylan digestibilities increased sharply with enzyme loading up to 25mg protein/g-glucan, after which the response varied depending on the pretreatment method. For a fixed level of enzyme loading, dilute sulfuric acid (DA), SO2, and Lime pretreatments exhibited higher digestibility than the soaking in aqueous ammonia (SAA) and ammonia fiber expansion (AFEX). Supplementation of Novozyme-188 to Spezyme-CP improved the 72h glucan digestibility only for the SAA treated samples. The effect of β-glucosidase supplementation was discernible only at the early phase of hydrolysis where accumulation of cellobiose and oligomers is significant. Addition of β-glucosidase increased the xylan digestibility of alkaline treated samples due to the β-xylosidase activity present in Novozyme-188. [Copyright &y& Elsevier]

Abstract: The US Department of Energy-funded Biomass Refining CAFI (Consortium for Applied Fundamentals and Innovation) project has developed leading pretreatment technologies for application to switchgrass and has evaluated their effectiveness in recovering sugars from the coupled operations of pretreatment and enzymatic hydrolysis. Key chemical and physical characteristics have been determined for pretreated switchgrass samples. Several analytical microscopy approaches utilizing instruments in the Biomass Surface Characterization Laboratory (BSCL) at the National Renewable Energy Laboratory (NREL) have been applied to untreated and CAFI-pretreated switchgrass samples. The results of this work have shown that each of the CAFI pretreatment approaches on switchgrass result in different structural impacts at the plant tissue, cellular, and cell wall levels. Some of these structural changes can be related to changes in chemical composition upon pretreatment. There are also apparently different structural mechanisms that are responsible for achieving the highest enzymatic hydrolysis sugar yields. [Copyright &y& Elsevier]

Abstract: Feedstock quality of switchgrass for biofuel production depends on many factors such as morphological types, geographic origins, maturity, environmental and cultivation parameters, and storage. We report variability in compositions and enzymatic digestion efficiencies for three cultivars of switchgrass (Alamo, Dacotah and Shawnee), grown and harvested at different locations and seasons. Saccharification yields of switchgrass processed by different pretreatment technologies (AFEX, dilute sulfuric acid, liquid hot water, lime, and soaking in aqueous ammonia) are compared in regards to switchgrass genotypes and harvest seasons. Despite its higher cellulose content per dry mass, Dacotah switchgrass harvested after wintering consistently gave a lower saccharification yield than the other two varieties harvested in the fall. The recalcitrance of upland cultivars and over-wintered switchgrass may require more severe pretreatment conditions. We discuss the key features of different pretreatment technologies and differences in switchgrass cultivars and harvest seasons on hydrolysis performance for the applied pretreatment methods. [Copyright &y& Elsevier]

Abstract: This work studied the benefits of adding different enzyme cocktails (cellulase, xylanase, β-glucosidase) to pretreated switchgrass. Pretreatment methods included ammonia fiber expansion (AFEX), dilute-acid (DA), liquid hot water (LHW), lime, lime+ball-milling, soaking in aqueous ammonia (SAA), and sulfur dioxide (SO2). The compositions of the pretreated materials were analyzed and showed a strong correlation between initial xylan composition and the benefits of xylanase addition. Adding xylanase dramatically improved xylan yields for SAA (+8.4%) and AFEX (+6.3%), and showed negligible improvement (0–2%) for the pretreatments with low xylan content (dilute-acid, SO2). Xylanase addition also improved overall yields with lime+ball-milling and SO2 achieving the highest overall yields from pretreated biomass (98.3% and 93.2%, respectively). Lime+ball-milling obtained an enzymatic yield of 92.3kg of sugar digested/kg of protein loaded. [Copyright &y& Elsevier]