The deconstruction of renewable biomass feedstocks into soluble sugars at low cost is a critical component of the biochemical conversion of biomass to fuels and chemicals. Providing low cost high concentration sugar syrups with low levels of chemicals and toxic inhibitors, at high process yields is essential for biochemical platform processes using pretreatment and enzymatic hydrolysis. In this work, we utilize a process consisting of deacetylation, followed by mechanical refining in a disc refiner (DDR) for the conversion of renewable biomass to low cost sugars at high yields and at high concentrations without a conventional chemical pretreatment step. The new process features a low temperature dilute alkaline deacetylation step followed by disc refining under modest levels of energy consumption. The proposed process was demonstrated using a commercial scale Andritz double disc refiner. Disc refined and deacetylated corn stover result in monomeric glucose yields of 78 to 84% and monomeric xylose yields of 71 to 77% after enzymatic hydrolysis at process-relevant solids and enzyme loadings. The glucose and xylose yields of the disc refined substrates in enzymatic hydrolysis are enhanced by 13% and 19%, respectively. Fermentation of the DDR substrates at 20% total solids with Z.mobilis utilized almost all sugars in 20hrs indicating the sugar hydrolyzate produced from the DDR process is highly fermentable due to low levels of chemical contaminants. The ethanol titer and ethanol process yield are approximately 70 g/L and 90% respectively. The proposed new process has been demonstrated using pilot scale deacetylation and disc refiners. The deacetylated and disc refined corn stover was rapidly deconstructed to monomeric sugars at 20% wt solids with enzymatic hydrolysis. High process sugar conversions were achieved, with high concentrations of monomeric sugars that exceeded 150 g/L. The sugar syrups produced were found to have low concentrations of known major fermentation inhibitors: acetic acid, furfural and HMF. The low levels of these fermentation inhibitors lead to high fermentation yields. The results suggest that this process is a very promising development for the nascent cellulosic biofuels industry.
Summary Conversion of nongrain biomass into liquid fuel is a sustainable approach to energy demands as global population increases. Previously, we showed that iron can act as a catalyst to enhance the degradation of lignocellulosic biomass for biofuel production. However, direct addition of iron catalysts to biomass pretreatment is diffusion‐limited, would increase the cost and complexity of biorefinery unit operations and may have deleterious environmental impacts. Here, we show a new strategy for in planta accumulation of iron throughout the volume of the cell wall where iron acts as a catalyst in the deconstruction of lignocellulosic biomass. We engineered CBM ‐ IBP fusion polypeptides composed of a carbohydrate‐binding module family 11 ( CBM 11) and an iron‐binding peptide ( IBP ) for secretion into Arabidopsis and rice cell walls. CBM ‐ IBP transformed Arabidopsis and rice plants show significant increases in iron accumulation and biomass conversion compared to respective controls. Further, CBM ‐ IBP rice shows a 35% increase in seed iron concentration and a 40% increase in seed yield in greenhouse experiments. CBM ‐ IBP rice potentially could be used to address iron deficiency, the most common and widespread nutritional disorder according to the World Health Organization.
1,4-@-D-Glucan glucohydrolase (exo-1 ,4-B-D-glUCOsidase) (EC 3.2.1.74)was isolated from growth supernatants of Torulopsis wickerhamii and was subjected to hydrodynamic, optical (CD), and kinetic analysis after purification to homogeneity by ammonium sulfate precipitation, size exclusion chromatography, ion exchange chromatography, and isopycnic banding centrifugation in cesium chloride.The last step was found to separate the enzyme from strongly associating, high molecular weight polysaccharide.Enzyme homogeneity was established by isoelectric focusing, sodium dodecyl sulfate-gel electrophoresis, and analytical high performance size exclusion chromatography using dual detection.The native exo-l,4-@-~-ghcosidase was found to be a dimer of 151,000 f 21,100 daltons by high performance size exclusion chromatography and 143,600 f 1,800 daltons by sedimentation equilibrium.The enzyme has a 12% linked carbohydrate content (mostly mannose) and no essential metal ions.Hydrolysis of p-nitrophenyl-@-D-glucopyranoside was found to be optimal at pH 4.25 and 50 "C.The enzyme was found to produce B-D-glucose from cellodextrins (indicating retention of anomeric configuration during hydrolysis) and demonstrated depolymerization from the non-reducing polymer terminus.The enzyme followed competitive type inhibition with p-nitrophenyl-@-D-glucopyranoside as substrate and demonstrated high values of Ki for D-glucose and D-cellobiose inhibition (190 and 230 mM, respectively).The exo-1,4-@-D-glucosidase was found to hydrolyze cellotetraose more rapidly than D-cellobiose and aryl-fi-D-glycosides more rapidly than all other substrates.Low levels of activity were found for the polymeric substrates 8glucan (yeast cell walls), Avicel, and Walseth cellulose.Although this enzyme demonstrates broad disaccharide substrate specificity, a characteristic common to 6-Dglucosidases from many sources, the ability to hydrolyze higher cellodextrins more rapidly than cellobiose renders this enzyme the first exo-1,4-/3-~-glucosidase purified from yeast.The production of ethanol from cellulose in abundant woody materials and agricultural wastes could be beneficial in relieving the strain on food grain stocks as well as in providing additional revenues from biomass utilization.How-
Pretreatment refers to the process unit operation that is responsible for disrupting the naturally resistant structure of lignocellulosic biomass to provide reactive intermediates, such as sugars and sugar degradation products and so on, to biochemical or thermochemical processes for production of biofuels and bioproducts. Various promising pretreatment technologies have been developed through employing bench-scale pretreatment systems to obtain data that lead to new models and new insights on mechanisms, facilitating the development and commercialization of low-cost pretreatment technologies. However, the ability to design and select these systems in conjunction with bioprocessing of biomass a priori is limited, and detailed mass and heat transfer data are vital for the assessment and scale-up of these technologies. Batch reactors, including sealed glass reactors, tubular reactors, mixed reactors, microwave reactors and steam reactors, as well as continuous pretreatment systems such as flowthrough systems, have been widely applied to establish a fundamental understanding of pretreatment mechanisms, to investigate kinetics of thermochemical biomass deconstruction reactions, to provide process simulations for techno-economical assessments, and to improve reactor designs. This chapter discusses the applications and characteristics of different bench-scale pretreatment systems, mass and heat transfer in these systems, and the selection of bench-scale pretreatment systems.
Two-stage dilute acid pretreatment followed by enzymatic cellulose hydrolysis is an effective method for obtaining high sugar yields from wood residues such as softwood forest thinnings. In the first-stage hydrolysis step, most of the hemicellulose is solubilized using relatively mild conditions. The soluble hemicellulosic sugars are recovered from the hydrolysate slurry by washing with water. The washed solids are then subjected to more severe hydrolysis conditions to hydrolyze approx 50% of the cellulose to glucose. The remaining cellulose can further be hydrolyzed with cellulase enzyme. Our process simulation indicates that the amount of water used in the hemicellulose recovery step has a significant impact on the cost of ethanol production. It is important to keep water usage as low as possible while maintaining relatively high recovery of soluble sugars. To achieve this objective, a proto-type pilot-scale continuous countercurrent screw extractor was evaluated for the recovery of hemicellulose from pretreated forest thinnings. Using the 274-cm (9-ft) long extractor, solubles recoveries of 98, 91, and 77% were obtained with liquid-to-insoluble solids (L/IS) ratios of 5.6, 3.4, and 2.1, respectively. An empirical equation was developed to predict the performance of the screw extractor. This equation predicts that soluble sugar recovery above 95% can be obtained with an L/IS ratio as low as 3.0.
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