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A structured understanding of cellobiohydrolase I binding to poplar lignin fractions after dilute acid pretreatment.

ABSTRACT: Cellulase adsorption to lignin is considered a cost barrier for bioethanol production; however, its detailed association mechanism is still not fully understood. In this study, two natural poplar variants with high and low sugar release performance were selected as the low and high recalcitrant raw materials (named L and H, respectively). Three different lignin fractions were extracted using ethanol, followed by p-dioxane and then cellulase treatment from the dilute acid pretreated poplar solids (fraction 1, 2, and 3, respectively).Each lignin fraction had different physicochemical properties. Ethanol-extracted lignin had the lowest weight average molecular weight, while the molecular weights for the other two lignin fractions were similar. 31P NMR analysis revealed that lignin fraction with higher molecular weight contained more aliphatic hydroxyl groups and less phenolic hydroxyl groups. Semi-quantitative analysis by 2D HSQC NMR indicated that the lignin fractions isolated from the natural variants had different contents of syringyl (S), guaiacyl (G) and interunit linkages. Lignin extracted by ethanol contained the largest amount of S units, the smallest amounts of G and p-hydroxybenzoate (PB) subunits, while the contents of these lignin subunits in the other two lignin fractions were similar. The lignin fraction obtained after cellulase treatment was primarily comprised of ?-O-4 linkages with small amounts of ?-5 and ?-? linkages. The binding strength of these three lignin fractions obtained by Langmuir equations were in the order of L1?>?L3?>?L2 for the low recalcitrance poplar and H1?>?H2?>?H3 for the high recalcitrance poplar.Overall, adsorption ability of lignin was correlated with the sugar release of poplar. Structural features of lignin were associated with its binding to CBH. For natural poplar variants, lignin fractions with lower molecular weight and polydispersity index (PDI) exhibited more CBH adsorption ability. Lignins with more phenolic hydroxyl groups had higher CBH binding strength. It was also found that lignin fractions with more condensed aromatics adsorbed more CBH likely attributed to stronger hydrophobic interactions.

SUBMITTER: Yao L

PROVIDER: S-EPMC5883885 | biostudies-other | 2018

REPOSITORIES: biostudies-other

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A structured understanding of cellobiohydrolase I binding to poplar lignin fractions after dilute acid pretreatment.

Yao Lan L Yoo Chang Geun CG Meng Xianzhi X Li Mi M Pu Yunqiao Y Ragauskas Arthur J AJ Yang Haitao H

Biotechnology for biofuels 20180404

<h4>Background</h4>Cellulase adsorption to lignin is considered a cost barrier for bioethanol production; however, its detailed association mechanism is still not fully understood. In this study, two natural poplar variants with high and low sugar release performance were selected as the low and high recalcitrant raw materials (named <i>L</i> and <i>H</i>, respectively). Three different lignin fractions were extracted using ethanol, followed by <i>p</i>-dioxane and then cellulase treatment from ...[more]

PMID: 29632555

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Project description:BackgroundPretreatment is a vital but expensive step in biomass biofuel production. Overall, most of this past effort has been directed at maximizing sugar yields from hemicellulose and cellulose through trials with different chemicals, operating conditions, and equipment configurations. Flowthrough pretreatment provides a promising platform to dissolution of lignocellulosic biomass to generate high yields of fermentable sugars and lignin for biofuels productions.ResultsDissolution of xylan, lignin, and cellulose from poplar wood were significantly enhanced by water-only and dilute acid (0.05% w/w, H2SO4) flowthrough pretreatment when the temperature was raised from 200°C to 280°C over a range of flow rates 10-62.5 mL/min, resulting in more than 98% solid removal. Up to 40% of original xylan was converted to xylose in the hydrolyzate and the rest xylan was solubilized into xylooligomers with negligible furfural formation. Up to 100% cellulose was removed into hydrolyzate with the highest glucose yield of 60% and low 5-hydroxymethylfurfural (5-HMF) formation. The maximal recovered insoluble lignin and soluble lignin were 98% and 15% of original lignin, respectively. In addition, enzymatic hydrolysis of pretreated whole slurries was characterized under various enzyme loadings with or without Bovine serum albumin (BSA) treatment. More than 90% glucose yield and 95% xylose yield were obtained from enzymatic hydrolysis of dilute acid pretreated whole slurries with 10 mg protein Ctec 2 with 2 mg Htec2/g glucan + xylan.ConclusionsNearly complete dissolution of whole biomass was realized through water-only and dilute acid flowthrough pretreatment under tested conditions. Temperature was considered as the most significant factor for cellulose degradation. The cellulose removal significantly increased as temperature reached 240°C for water-only and 220°C for dilute acid. Dilute acid pretreatment resulted in higher yields of recovered xylan and cellulose as monomeric sugars in the hydrolyzate than that for water-only pretreatment. Enzymes readily hydrolyzed the degraded cellulose and xylooligomers in pretreatment hydrolysate. Results suggested that kinetics controlled the flowthrough pretreatment of biomass dissolution, which was also affected by flow rate to certain extent.

Project description:BackgroundIn this study, the dilute maleic acid pretreatment of wheat straw is optimized, using pretreatment time, temperature and maleic acid concentration as design variables. A central composite design was applied to the experimental set up. The response factors used in this study are: (1) glucose benefits from improved enzymatic digestibility of wheat straw solids; (2) xylose benefits from the solubilization of xylan to the liquid phase during the pretreatment; (3) maleic acid replenishment costs; (4) neutralization costs of pretreated material; (5) costs due to furfural production; and (6) heating costs of the input materials. For each response factor, experimental data were fitted mathematically. After data translation to euro/Mg dry straw, determining the relative contribution of each response factor, an economic optimization was calculated within the limits of the design variables.ResultsWhen costs are disregarded, an almost complete glucan conversion to glucose can be reached (90% from solids, 7%-10% in liquid), after enzymatic hydrolysis. During the pretreatment, up to 90% of all xylan is converted to monomeric xylose. Taking cost factors into account, the optimal process conditions are: 50 min at 170 degrees C, with 46 mM maleic acid, resulting in a yield of 65 euro/Mg (megagram = metric ton) dry straw, consisting of 68 euro/Mg glucose benefits (from solids: 85% of all glucan), 17 euro/Mg xylose benefits (from liquid: 80% of all xylan), 17 euro/Mg maleic acid costs, 2.0 euro/Mg heating costs and 0.68 euro/Mg NaOH costs. In all but the most severe of the studied conditions, furfural formation was so limited that associated costs are considered negligible.ConclusionsAfter the dilute maleic acid pretreatment and subsequent enzymatic hydrolysis, almost complete conversion of wheat straw glucan and xylan is possible. Taking maleic acid replenishment, heating, neutralization and furfural formation into account, the optimum in the dilute maleic acid pretreatment of wheat straw in this study is 65 euro/Mg dry feedstock. This is reached when process conditions are: 50 min at 170 degrees C, with a maleic acid concentration of 46 mM. Maleic acid replenishment is the most important of the studied cost factors.

Project description:BackgroundHighly efficient enzymatic saccharification of pretreated lignocellulose is a key step in achieving lignocellulosic biorefinery. Cellobiohydrolase I (Cel7A) secreted by Trichoderma reesei is an industrially used cellulase that possesses carbohydrate-binding module 1 (TrCBM1) at the C-terminal domain. The nonproductive binding of TrCBM1 to lignin significantly decreases the enzymatic saccharification efficiency and increases the cost of biomass conversion because of the additionally required enzymes. Understanding the interaction mechanism between lignin and TrCBM1 is essential for realizing a cost-effective biofuel production; however, the binding sites in lignin have not been clearly elucidated.ResultsThree types of 13C-labeled β-O-4 lignin oligomer models were synthesized and characterized. The 2D 1H-13C heteronuclear single-quantum correlation (HSQC) spectra of the 13C-labeled lignin models confirmed that the three types of the 13C labels were correctly incorporated in the (1) aromatic rings and β positions, (2) α positions, and (3) methoxy groups, respectively. The TrCBM1-binding sites in lignin were analyzed by observing NMR chemical shift perturbations (CSPs) using the synthetic 13C-labeled β-O-4 lignin oligomer models. Obvious CSPs were observed in signals from the aromatic regions in oligomers bound to TrCBM1, whereas perturbations in the signals from aliphatic regions and methoxy groups were insignificant. These findings indicated that hydrophobic interactions and π-π stacking were dominating factors in nonproductive binding. The synthetic lignin models have two configurations whose terminal units were differently aligned and donated C(I) and C(II). The C(I) ring showed remarkable perturbation compared with the C(II), which indicated that the binding of TrCBM1 was markedly affected by the configuration of the lignin models. The long-chain lignin models (degree of polymerization (DP) 4.16-4.70) clearly bound to TrCBM1. The interactions of TrCBM1 with the short-chain lignin models (DP 2.64-3.12) were insignificant, indicating that a DP greater than 4 was necessary for TrCBM1 binding.ConclusionThe CSP analysis using 13C-labeled β-O-4 lignin oligomer models enabled the identification of the TrCBM1 binding sites in lignins at the atomic level. This specific interaction analysis will provide insights for new molecular designs of cellulase having a controlled affinity to cellulose and lignin for a cost-effective biorefinery process.

Project description:BackgroundDilute acid pretreatment is a promising process technology for the deconstruction of low-lignin lignocellulosic biomass, capable of producing high yields of hemicellulosic sugars and enhancing enzymatic yields of glucose as part of a biomass-to-biofuels process. However, while it has been extensively studied, most work has historically been conducted at relatively high acid concentrations of 1 - 4% (weight/weight). Reducing the effective acid loading in pretreatment has the potential to reduce chemical costs both for pretreatment and subsequent neutralization. Additionally, if acid loadings are sufficiently low, capital requirements associated with reactor construction may be significantly reduced due to the relaxation of requirements for exotic alloys. Despite these benefits, past efforts have had difficulty obtaining high process yields at low acid loadings without supplementation of additional unit operations, such as mechanical refining.ResultsRecently, we optimized the dilute acid pretreatment of deacetylated corn stover at low acid loadings in a 1-ton per day horizontal pretreatment reactor. This effort included more than 25 pilot-scale pretreatment experiments executed at reactor temperatures ranging from 150 - 170°C, residence times of 10 - 20 minutes and hydrolyzer sulfuric acid concentrations between 0.15 - 0.30% (weight/weight). In addition to characterizing the process yields achieved across the reaction space, the optimization identified a pretreatment reaction condition that achieved total xylose yields from pretreatment of 73.5% ± 1.5% with greater than 97% xylan component balance closure across a series of five runs at the same condition. Feedstock reactivity at this reaction condition after bench-scale high solids enzymatic hydrolysis was 77%, prior to the inclusion of any additional conversion that may occur during subsequent fermentation.ConclusionsThis study effectively characterized a range of pretreatment reaction conditions using deacetylated corn stover at low acid loadings and identified an optimum reaction condition was selected and used in a series of integrated pilot scale cellulosic ethanol production campaigns. Additionally, several issues exist to be considered in future pretreatment experiments in continuous reactor systems, including the formation of char within the reactor, as well as practical issues with feeding herbaceous feedstock into pressurized systems.

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A structured understanding of cellobiohydrolase I binding to poplar lignin fractions after dilute acid pretreatment.

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A structured understanding of cellobiohydrolase I binding to poplar lignin fractions after dilute acid pretreatment.

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