Abstract Background In converting biomass to bioethanol, pretreatment is a key step intended to render cellulose more amenable and accessible to cellulase enzymes and thus increase glucose yields. In this study, four cellulose samples with different degrees of polymerization and crystallinity indexes were subjected to aqueous sodium hydroxide and anhydrous liquid ammonia treatments. The effects of the treatments on cellulose crystalline structure were studied, in addition to the effects on the digestibility of the celluloses by a cellulase complex. Results From X-ray diffractograms and nuclear magnetic resonance spectra, it was revealed that treatment with liquid ammonia produced the cellulose III I allomorph; however, crystallinity depended on treatment conditions. Treatment at a low temperature (25°C) resulted in a less crystalline product, whereas treatment at elevated temperatures (130°C or 140°C) gave a more crystalline product. Treatment of cellulose I with aqueous sodium hydroxide (16.5 percent by weight) resulted in formation of cellulose II, but also produced a much less crystalline cellulose. The relative digestibilities of the different cellulose allomorphs were tested by exposing the treated and untreated cellulose samples to a commercial enzyme mixture (Genencor-Danisco; GC 220). The digestibility results showed that the starting cellulose I samples were the least digestible (except for corn stover cellulose, which had a high amorphous content). Treatment with sodium hydroxide produced the most digestible cellulose, followed by treatment with liquid ammonia at a low temperature. Factor analysis indicated that initial rates of digestion (up to 24 hours) were most strongly correlated with amorphous content. Correlation of allomorph type with digestibility was weak, but was strongest with cellulose conversion at later times. The cellulose III I samples produced at higher temperatures had comparable crystallinities to the initial cellulose I samples, but achieved higher levels of cellulose conversion, at longer digestion times. Conclusions Earlier studies have focused on determining which cellulose allomorph is the most digestible. In this study we have found that the chemical treatments to produce different allomorphs also changed the crystallinity of the cellulose, and this had a significant effect on the digestibility of the substrate. When determining the relative digestibilities of different cellulose allomorphs it is essential to also consider the relative crystallinities of the celluloses being tested.
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.
To develop a direct microbial sugar conversion platform for the production of lipids, drop-in fuels and chemicals from cellulosic biomass substrate, we chose Yarrowia lipolytica as a viable demonstration strain. Y. lipolytica is known to accumulate lipids intracellularly and is capable of metabolizing sugars to produce lipids; however, it lacks the lignocellulose-degrading enzymes needed to break down biomass directly. While research is continuing on the development of a Y. lipolytica strain able to degrade cellulose, in this study, we present successful expression of several xylanases in Y. lipolytica. The XynII and XlnD expressing Yarrowia strains exhibited an ability to grow on xylan mineral plates. This was shown by Congo Red staining of halo zones on xylan mineral plates. Enzymatic activity tests further demonstrated active expression of XynII and XlnD in Y. lipolytica. Furthermore, synergistic action in converting xylan to xylose was observed when XlnD acted in concert with XynII. The successful expression of these xylanases in Yarrowia further advances us toward our goal to develop a direct microbial conversion process using this organism.
Lignocellulosic biomass is a renewable, naturally mass-produced form of stored solar energy. Thermochemical pretreatment processes have been developed to address the challenge of biomass recalcitrance, however the optimization, cost reduction, and scalability of these processes remain as obstacles to the adoption of biofuel production processes at the industrial scale. In this study, we demonstrate that the type of reactor in which pretreatment is carried out can profoundly alter the micro- and nanostructure of the pretreated materials and dramatically affect the subsequent efficiency, and thus cost, of enzymatic conversion of cellulose. Multi-scale microscopy and quantitative image analysis was used to investigate the impact of different biomass pretreatment reactor configurations on plant cell wall structure. We identify correlations between enzymatic digestibility and geometric descriptors derived from the image data. Corn stover feedstock was pretreated under the same nominal conditions for dilute acid pretreatment (2.0 wt% H2SO4, 160°C, 5 min) using three representative types of reactors: ZipperClave® (ZC), steam gun (SG), and horizontal screw (HS) reactors. After 96 h of enzymatic digestion, biomass treated in the SG and HS reactors achieved much higher cellulose conversions, 88% and 95%, respectively, compared to the conversion obtained using the ZC reactor (68%). Imaging at the micro- and nanoscales revealed that the superior performance of the SG and HS reactors could be explained by reduced particle size, cellular dislocation, increased surface roughness, delamination, and nanofibrillation generated within the biomass particles during pretreatment. Increased cellular dislocation, surface roughness, delamination, and nanofibrillation revealed by direct observation of the micro- and nanoscale change in accessibility explains the superior performance of reactors that augment pretreatment with physical energy.
5-hydroxymethylfurfural (HMF) and furfural are promising platform molecules which can be converted to fuel intermediates and hydrocarbon fuels. Herein, one-step, direct conversion of the carbohydrates in never treated biomass to HMF and furfural in a microwave reactor was investigated. The influence of reaction temperature, time, feedstock, addition of Lewis and Brønsted acids, solvent and solvent to aqueous ratio were studied to find the optimum process conditions to maximize the yields of furfurals. The reaction conditions of 200 °C for 5 min with 33 mM HCl and 8 mM AlCl3 using a dioxane/water (4:1) miscible solvent system were found to be the most conducive for direct dehydration of the glucan and hemicelluloses present in untreated poplar wood to furfural and HMF in yields of 91 and 69%, respectively. Furfural could be easily produced in yields greater than 80%, and once formed, it was found to be stable under the various process conditions explored. In contrast, HMF yields varied with both reaction time and temperature, as it readily underwent rehydration to levulinic acid in yields approaching 25–30% at increased reaction severity. The additions of both Brønsted (HCl) and Lewis (AlCl3) acids in catalytic amounts, i.e., 33 and 8 mM, respectively, were required to maximize the yield of furfurals. Reactions were also performed on other feedstocks (corn stover, switchgrass, loblolly pine, and pure cellulose) to investigate the effect of feedstock type on furfurals yields. Results showed that HMF and furfural yields were primarily dependent on hexosan and pentosan loading regardless of the type of feedstock. In addition, biomass physical features, such as crystallinity or the presence of other biomass constituents, such as lignin, had little influence on furfurals yields.
Dilute acid pretreatment is a promising pretreatment technology for the biochemical production of ethanol from lignocellulosic biomass. During dilute acid pretreatment, xylan depolymerizes to form soluble xylose monomers and oligomers. Because the xylan found in nature is highly acetylated, the formation of xylose monomers requires two steps: 1) cleavage of the xylosidic bonds, and 2) cleavage of covalently bonded acetyl ester groups.In this study, we show that the latter may be the rate limiting step for xylose monomer formation. Furthermore, acetyl groups are also found to be a cause of biomass recalcitrance and hydrolyzate toxicity. While the removal of acetyl groups from native corn stover by alkaline de-esterification prior to pretreatment improves overall process yields, the exact impact is highly dependent on the corn stover variety in use. Xylose monomer yields in pretreatment generally increases by greater than 10%. Compared to pretreated corn stover controls, the deacetylated corn stover feedstock is approximately 20% more digestible after pretreatment. Finally, by lowering hydrolyzate toxicity, xylose utilization and ethanol yields are further improved during fermentation by roughly 10% and 7%, respectively. In this study, several varieties of corn stover lots were investigated to test the robustness of the deacetylation-pretreatment-saccharification-fermentation process.Deacetylation shows significant improvement on glucose and xylose yields during pretreatment and enzymatic hydrolysis, but it also reduces hydrolyzate toxicity during fermentation, thereby improving ethanol yields and titer. The magnitude of effect is dependent on the selected corn stover variety, with several varieties achieving improvements of greater than 10% xylose yield in pretreatment, 20% glucose yield in low solids enzymatic hydrolysis and 7% overall ethanol yield.
Xylose reversion reactions to form xylooligomers represent a potentially important mechanism of sugar loss during dilute acid pretreatment of biomass. We have conducted a study to identify the products that result from these reactions and to determine the kinetics of their formation. A major obstacle is that there are few commercial standards available for xylose disaccharides, which are essential for the identification and quantification of the xylose reversion products formed during these reactions. To overcome this obstacle, we have used GC/MS and NMR analysis of xylose disaccharides isolated by preparative HPLC to identify the reaction products. At the xylose concentration we used (300 g L–1), only xylose disaccharides were observed. As with glucose reversion reactions [Pilath, H. M.; et al. J. Agric. Food Chem. 2010, 58, 6131], the disaccharides contained linkages that involved the anomeric carbon atom of one of the sugar monomers. Eight out of the nine possible disaccharides, including alpha and beta anomers, were observed. Whereas the GC/MS allowed for the identification of the linkages, NMR was needed to distinguish between the α and β isomers of the disaccharides. The kinetics of combined xylose disaccharide formation was measured using HPLC. Arrhenius parameters for the rates of disaccharide formation were calculated by fitting the data to a simple model.
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