Lignin content in natural Populus variants affects sugar release.

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.

Application of high throughput pretreatment and co-hydrolysis system to thermochemical pretreatment. Part 1: dilute acid.

Because conventional approaches for evaluating sugar release from the coupled operations of pretreatment and enzymatic hydrolysis are extremely time and material intensive, high throughput (HT) pretreatment and enzymatic hydrolysis systems have become vital for screening large numbers of lignocellulosic biomass samples to identify feedstocks and/or processing conditions that significantly improve performance and lower costs. Because dilute acid pretreatment offers many important advantages in rendering biomass highly susceptible to subsequent enzymatic hydrolysis, a high throughput pretreatment and co-hydrolysis (HTPH) approach was extended to employ dilute acid as a tool to screen for enhanced performance. First, a single-step neutralization and buffering method was developed to allow effective enzymatic hydrolysis of the whole pretreated slurry. Switchgrass and poplar were then pretreated with 0.5% and 1% acid loadings at a 5% solids concentration, the resulting slurry conditioned with the buffering approach, and the entire mixture enzymatically hydrolyzed. The resulting sugar yields demonstrated that single-step neutralizing and buffering was capable of adjusting the pH as needed for enzymatic saccharification, as well as overcoming enzyme inhibition by compounds released in pretreatment. In addition, the effects of pretreatment conditions and biomass types on susceptibility of pretreated substrates to enzymatic conversion were clearly discernible, demonstrating the method to be a useful extension of HTPH systems.

Application of high throughput pretreatment and co-hydrolysis system to thermochemical pretreatment. Part 2: Dilute alkali.

High throughput pretreatment (HTPH) and enzymatic hydrolysis systems are now vital for screening large numbers of biomass samples to investigate biomass recalcitrance over various pretreatment and enzymatic hydrolysis conditions. Although hydrothermal pretreatment is currently being employed in most high throughput applications, thermochemical pretreatment at low and high pH conditions can offer additional insights to better understand the roles of hemicellulose and lignin, respectively, in defining biomass recalcitrance. Thus, after successfully applying the HTPH approach to dilute acid pretreatment [Gao et al. (2012) Biotechnol. Bioeng. 110(3): 754-762], extension to dilute alkali pretreatment was also achieved using a similar single-step neutralization and buffering concept. In the latter approach, poplar and switchgrass were pretreated with 1 wt% sodium hydroxide at 120°C for different reaction times. Following pretreatment, an H2Cit⁻/HCit²â» buffer with a pH of 4.5 was used to condition the pretreatment slurry to a pH range of 4.69-4.89, followed by enzymatic hydrolysis for 72 h of the entire mixture. Sugar yields showed different trends for poplar and switchgrass with increases in pretreatment times, demonstrating the method provided a clearly discernible screening tool at alkali conditions. This method was then applied to selected Populus tremuloides samples to follow ring-by-ring sugar release patterns. Observed variations were compared to results from hydrothermal pretreatments, providing new insights in understanding the influence of biomass structural differences on recalcitrance.

Small-scale and automatable high-throughput compositional analysis of biomass.

Conventional wet chemistry methods to determine biomass composition are labor- and time-intensive and require larger amounts of biomass (300 mg) than is often available. To overcome these limitations and to support a high-throughput pretreatment and hydrolysis (HTPH) screening system, this article reports on the development of a downscaled biomass compositional analysis that is based on conventional wet chemistry techniques but is scaled down by a factor of 100 to use significantly less material. The procedure is performed in readily available high-performance liquid chromatography vials and can be automated to reduce operator input and increase throughput. Comparison of the compositional analyses of three biomasses determined by the downscaled approach to those obtained by conventional methods showed that the downscaled method measured statistically identical carbohydrate compositions as standard procedures and also can provide reasonable estimates of lignin and ash contents. These results demonstrate the validity of the downscaled procedure for measuring biomass composition to enable the calculation of sugar yields and determination of trends in sugar release behavior in HTPH screening studies.

Engineering of a high-throughput screening system to identify cellulosic biomass, pretreatments, and enzyme formulations that enhance sugar release.

The recalcitrance of cellulosic biomass, the only abundant, sustainable feedstock for making liquid fuels, is a primary obstacle to low cost biological processing, and development of more easily converted plants and more effective enzymes would be of great benefit. Because no single parameter describes recalcitrance, superior variants can only be identified by measuring sugar release from plants subjected to pretreatment and enzymatic hydrolysis. However, genetic modifications of plants coupled with molecular engineering of deconstruction proteins and definition of pretreatment conditions create a very large sample set, and previous methods for biomass pretreatment at elevated temperatures and pressures prevented use of a fully integrated high-throughput (HTP) screening pipeline. Herein, we report on the engineering of a novel HTP pretreatment system employing a 96 well-plate format that withstands extreme pretreatment conditions for rapid screening of biomass-enzyme-pretreatment combinations. This includes the development of new approaches to steam heating and water quenching the system that result in much faster heat up and cool down than previously possible and show consistent temperature histories across the multiwell plate. Coupled pretreatment and enzymatic hydrolysis performance of the well plate pretreatment system is shown to be consistent among the many wells in the device and also with performance of conventional tubular reactors.

How chip size impacts steam pretreatment effectiveness for biological conversion of poplar wood into fermentable sugars.

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.

Changes in composition and sugar release across the annual rings of Populus wood and implications on recalcitrance.

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.

Composition and hydrothermal pretreatment and enzymatic saccharification performance of grasses and legumes from a mixed-species prairie.

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.

Co-hydrolysis of hydrothermal and dilute acid pretreated populus slurries to support development of a high-throughput pretreatment system.

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.