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

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

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

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

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

Comparison of the Effectiveness of a Fluidized Sand Bath and a Steam Chamber for Reactor Heating

Both fluidized sand baths and steam chambers have been used to heat laboratory reactors, in particular for studies of biomass pretreatment. In this study, several aspects of the heating performance of these devices were compared: time to heat reactors to reaction temperature, the stability of reactor temperature, and the convection coefficient. The convection coefficient was determined using correlations and multiple analyses of empirical data. On the basis of the results presented in this study, the steam chamber can heat reactors to temperature in a tenth of the time sand baths can, can maintain a more stable temperature during pretreatment, and has a convection coefficient one to two magnitudes greater than that of the sand bath. Therefore if heat transfer is critical, a steam chamber is advantageous

Co-hydrolysis of hydrothermal and dilute acid pretreated <it>populus </it>slurries to support development of a high-throughput pretreatment system

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.</p

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

Comparison of the Effectiveness of a Fluidized Sand Bath and a Steam Chamber for Reactor Heating

Both fluidized sand baths and steam chambers have been used to heat laboratory reactors, in particular for studies of biomass pretreatment. In this study, several aspects of the heating performance of these devices were compared: time to heat reactors to reaction temperature, the stability of reactor temperature, and the convection coefficient. The convection coefficient was determined using correlations and multiple analyses of empirical data. On the basis of the results presented in this study, the steam chamber can heat reactors to temperature in a tenth of the time sand baths can, can maintain a more stable temperature during pretreatment, and has a convection coefficient one to two magnitudes greater than that of the sand bath. Therefore if heat transfer is critical, a steam chamber is advantageous