Search

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

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.

Abstract Background Effective enzymatic hydrolysis of lignocellulosic biomass benefits from lignin removal, relocation, and/or modification during hydrothermal pretreatment. Phase transition, depolymerization/repolymerization, and solubility effects may all influence these lignin changes. To better understand how lignin is altered, Populus trichocarpa x P. deltoides wood samples and cellulolytic enzyme lignin (CEL) isolated from P. trichocarpa x P. deltoides were subjected to batch and flowthrough pretreatments. The residual solids and liquid hydrolysate were characterized by gel permeation chromatography, heteronuclear single quantum coherence NMR, compositional analysis, and gas chromatography–mass spectrometry. Results Changes in the structure of the solids recovered after the pretreatment of CEL and the production of aromatic monomers point strongly to depolymerization and condensation being primary mechanisms for lignin extraction and redeposition. The differences in lignin removal and phenolic compound production from native P. trichocarpa x P. deltoides and CEL suggested that lignin-carbohydrate interactions increased lignin extraction and the extractability of syringyl groups relative to guaiacyl groups. Conclusions These insights into delignification during hydrothermal pretreatment point to desirable pretreatment strategies and plant modifications. Because depolymerization followed by repolymerization appears to be the dominant mode of lignin modification, limiting the residence time of depolymerized lignin moieties in the bulk liquid phase should reduce lignin content in pretreated biomass. In addition, the increase in lignin removal in the presence of polysaccharides suggests that increasing lignin-carbohydrate cross-links in biomass would increase delignification during pretreatment.

Abstract 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 Background Mixtures of prairie species (mixed prairie species; MPS) have been proposed to offer important advantages as a feedstock for sustainable production of fuels and chemicals. Therefore, understanding the performance in hydrothermal pretreatment and enzymatic hydrolysis of select species harvested from a mixed prairie is valuable in selecting these components for such applications. This study examined composition and sugar release from the most abundant components of a plot of MPS: a C3 grass (Poa pratensis), a C4 grass (Schizachyrium scoparium), and a legume (Lupinus perennis). Results from this study provide a platform to evaluate differences between grass and leguminous species, and the factors controlling their recalcitrance to pretreatment and enzymatic hydrolysis. Results Significant differences were found between the grass and leguminous species, and between the individual anatomical components that influence the recalcitrance of MPS. We found that both grasses contained higher levels of sugars than did the legume, and also exhibited higher sugar yields as a percentage of the maximum possible from combined pretreatment and enzymatic hydrolysis. Furthermore, particle size, acid-insoluble residue (AcIR), and xylose removal were not found to have a direct significant effect on glucan digestibility for any of the species tested, whereas anatomical composition was a key factor in both grass and legume recalcitrance, with the stems consistently exhibiting higher recalcitrance than the other anatomical fractions. Conclusions The prairie species tested in this study responded well to hydrothermal pretreatment and enzymatic saccharification. Information from this study supports recommendations as to which plant types and species are more desirable for biological conversion in a mixture of prairie species, in addition to identifying fractions of the plants that would most benefit from genetic modification or targeted growth.

Abstract Background The BioEnergy Science Center (BESC) developed a high-throughput screening method to rapidly identify low-recalcitrance biomass variants. Because the customary separation and analysis of liquid and solids between pretreatment and enzymatic hydrolysis used in conventional analyses is slow, labor-intensive and very difficult to automate, a streamlined approach we term 'co-hydrolysis' was developed. In this method, the solids and liquid in the pretreated biomass slurry are not separated, but instead hydrolysis is performed by adding enzymes to the whole pretreated slurry. The effects of pretreatment method, severity and solids loading on co-hydrolysis performance were investigated. Results For hydrothermal pretreatment at solids concentrations of 0.5 to 2%, high enzyme protein loadings of about 100 mg/g of substrate (glucan plus xylan) in the original poplar wood achieved glucose and xylose yields for co-hydrolysis that were comparable with those for washed solids. In addition, although poplar wood sugar yields from co-hydrolysis at 2% solids concentrations fell short of those from hydrolysis of washed solids after dilute sulfuric acid pretreatment even at high enzyme loadings, pretreatment at 0.5% solids concentrations resulted in similar yields for all but the lowest enzyme loading. Conclusions Overall, the influence of severity on susceptibility of pretreated substrates to enzymatic hydrolysis was clearly discernable, showing co-hydrolysis to be a viable approach for identifying plant-pretreatment-enzyme combinations with substantial advantages for sugar production.

Abstract 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 Background The BioEnergy Science Center (BESC) developed a high-throughput screening method to rapidly identify low-recalcitrance biomass variants. Because the customary separation and analysis of liquid and solids between pretreatment and enzymatic hydrolysis used in conventional analyses is slow, labor-intensive and very difficult to automate, a streamlined approach we term 'co-hydrolysis' was developed. In this method, the solids and liquid in the pretreated biomass slurry are not separated, but instead hydrolysis is performed by adding enzymes to the whole pretreated slurry. The effects of pretreatment method, severity and solids loading on co-hydrolysis performance were investigated. Results For hydrothermal pretreatment at solids concentrations of 0.5 to 2%, high enzyme protein loadings of about 100 mg/g of substrate (glucan plus xylan) in the original poplar wood achieved glucose and xylose yields for co-hydrolysis that were comparable with those for washed solids. In addition, although poplar wood sugar yields from co-hydrolysis at 2% solids concentrations fell short of those from hydrolysis of washed solids after dilute sulfuric acid pretreatment even at high enzyme loadings, pretreatment at 0.5% solids concentrations resulted in similar yields for all but the lowest enzyme loading. Conclusions Overall, the influence of severity on susceptibility of pretreated substrates to enzymatic hydrolysis was clearly discernable, showing co-hydrolysis to be a viable approach for identifying plant-pretreatment-enzyme combinations with substantial advantages for sugar production.

Abstract 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.

Background: Hydrothermal pretreatment using liquid hot water (LHW) is capable of substantially reducing the cell wall recalcitrance of lignocellulosic biomass. It enhances the saccharification of polysaccharides, particularly cellulose, into glucose with relatively low capital required. Due to the close association with biomass recalcitrance, the structural change of the components of lignocellulosic materials during the pretreatment is crucial to understand pretreatment chemistry and advance the bio-economy. Although the LHW pretreatment has been extensively applied and studied, the molecular structural alteration during pretreatment and its significance to reduced recalcitrance have not been well understood. Results: We investigated the effects of LHW pretreatment with different severity factors (log R0) on the structural changes of fast-grown poplar (Populus trichocarpa). With the severity factor ranging from 3.6 to 4.2, LHW pretreatment resulted in a substantial xylan solubilization by 50-77% (w/w, dry matter). The molecular weights of the remained hemicellulose in pretreated solids also have been significantly reduced by 63-75% corresponding to LHW severity factor from 3.6 to 4.2. In addition, LHW had a considerable impact on the cellulose structure. The cellulose crystallinity increased 6-9%, whereas its degree of polymerization decreased 35-65% after pretreatment. We found that the pretreatment severity had an empirical linear correlation with the xylan solubilization (R2 = 0.98, r = + 0.99), hemicellulose molecular weight reduction (R2 = 0.97, r = - 0.96 and R2 = 0.93, r = - 0.98 for number-average and weightaverage degree of polymerization, respectively), and cellulose crystallinity index increase (R2 = 0.98, r = + 0.99). The LHW pretreatment also resulted in small changes in lignin structure such as decrease of β-O-4' ether linkages and removal of cinnamyl alcohol end group and acetyl group, while the S/G ratio of lignin in LHW pretreated poplar residue remained no significant change compared with the untreated poplar. Conclusions: This study revealed that the solubilization of xylan, the reduction of hemicellulose molecular weights and cellulose degree of polymerization, and the cleavage of alkyl-aryl ether bonds in lignin resulted from LHW pretreatment are critical factors associated with reduced cell wall recalcitrance. The chemical-structural changes of the three major components, cellulose, lignin, and hemicellulose, during LHW pretreatment provide useful and fundamental information of factors governing feedstock recalcitrance during hydrothermal pretreatment. [ABSTRACT FROM AUTHOR]

Background Obtaining a better understanding of the complex mechanisms occurring during lignocellulosic deconstruction is critical to the continued growth of renewable biofuel production. A key step in bioethanol production is thermochemical pretreatment to reduce plant cell wall recalcitrance for downstream processes. Previous studies of dilute acid pretreatment (DAP) have shown significant changes in cellulose ultrastructure that occur during pretreatment, but there is still a substantial knowledge gap with respect to the influence of lignin on these cellulose ultra structural changes. This study was designed to assess how the presence of lignin influences DAP-induced changes in cellulose ultra structure, which might ultimately have large implications with respect to enzymatic deconstruction efforts. Results Native, untreated hybrid poplar (Populus trichocarpa x Populus deltoids) samples and a partially delignified poplar sample (facilitated by acidic sodium chlorite pulping) were separately pretreated with dilute sulfuric acid (0.10 M) at 160°C for 15 minutes and 35 minutes, respectively . Following extensive characterization, the partially delignified biomass displayed more significant changes in cellulose ultra structure following DAP than the native untreated biomass. With respect to the native untreated poplar, delignified poplar after DAP (in which approximately 40% lignin removal occurred) experienced: increased cellulose accessibility indicated by increased Simons' stain (orange dye) adsorption from 21.8 to 72.5 mg/g, decreased cellulose weight-average degree of polymerization (DPw) from 3087 to 294 units, and increased cellulose crystallite size from 2.9 to 4.2 nm. These changes following DAP ultimately increased enzymatic sugar yield from 10 to 80%. Conclusions Overall, the results indicate a strong influence of lignin content on cellulose ultra structural changes occurring during DAP. With the reduction of lignin content during DAP, the enlargement of cellulose micro fibril dimensions and crystallite size becomes more apparent. Further, this enlargement of cellulose micro fibril dimensions is attributed to specific processes, including the co-crystallization of crystalline cellulose driven by irreversible inter chain hydrogen bonding (similar to hornification) and/or cellulose annealing that converts amorphous cellulose to paracrystalline and crystalline cellulose. Essentially, lignin acts as a barrier to prevent cellulose crystallinity increase and cellulose fibril coalescence during DAP. [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]

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]

Background: The recalcitrance of cellulosic biomass is widely recognized as a key barrier to cost-effective biological processing to fuels and chemicals, but the relative impacts of physical, chemical and genetic interventions to improve biomass processing singly and in combination have yet to be evaluated systematically. Solubilization of plant cell walls can be enhanced by non-biological augmentation including physical cotreatment and thermochemical pretreatment, the choice of biocatalyst, the choice of plant feedstock, genetic engineering of plants, and choosing feedstocks that are less recalcitrant natural variants. A two-tiered combinatoric investigation of lignocellulosic biomass deconstruction was undertaken with three biocatalysts (Clostridium thermocellum, Caldicellulosiruptor bescii, Novozymes Cellic® Ctec2 and Htec2), three transgenic switchgrass plant lines (COMT, MYB4, GAUT4) and their respective nontransgenic controls, two Populus natural variants, and augmentation of biological attack using either mechanical cotreatment or cosolvent-enhanced lignocellulosic fractionation (CELF) pretreatment. Results: In the absence of augmentation and under the conditions tested, increased total carbohydrate solubilization (TCS) was observed for 8 of the 9 combinations of switchgrass modifications and biocatalysts tested, and statistically significant for five of the combinations. Our results indicate that recalcitrance is not a trait determined by the feedstock only, but instead is coequally determined by the choice of biocatalyst. TCS with C. thermocellum was significantly higher than with the other two biocatalysts. Both CELF pretreatment and cotreatment via continuous ball milling enabled TCS in excess of 90%. Conclusion: Based on our results as well as literature studies, it appears that some form of non-biological augmentation will likely be necessary for the foreseeable future to achieve high TCS for most cellulosic feedstocks. However, our results show that this need not necessarily involve thermochemical processing, and need not necessarily occur prior to biological conversion. Under the conditions tested, the relative magnitude of TCS increase was augmentation > biocatalyst choice > plant choice > plant modification > plant natural variants. In the presence of augmentation, plant modification, plant natural variation, and plant choice exhibited a small, statistically non-significant impact on TCS. [ABSTRACT FROM AUTHOR]