Stability of commercial glucanase and β-glucosidase preparations under hydrolysis conditions

The cost of enzymes makes enzymatic hydrolysis one of the most expensive steps in the production of lignocellulosic ethanol. Diverse studies have used commercial enzyme cocktails assuming that change in total protein concentration during hydrolysis was solely due to adsorption of endo- and exoglucanases onto the substrate. Given the sensitivity of enzymes and proteins to media conditions this assumption was tested by evaluating and modeling the protein concentration of commercial cocktails at hydrolysis conditions. In the absence of solid substrate, the total protein concentration of a mixture of Celluclast 1.5 L and Novozyme 188 decreased by as much as 45% at 50 °C after 4 days. The individual cocktails as well as a mixture of both were stable at 20 °C. At 50 °C, the protein concentration of Celluclast 1.5 was relatively constant but Novozyme 188 decreased by as much as 77%. It was hypothesized that Novozyme 188 proteins suffer a structural change at 50 °C which leads to protein aggregation and precipitation. Lyophilized β-glucosidase (P-β-glucosidase) at 50 °C exhibited an aggregation rate which was successfully modeled using first order kinetics (R2 = 0.97). By incorporating the possible presence of chaperone proteins in Novozyme 188, the protein aggregation observed for this cocktail was successfully modeled (R2 = 0.96). To accurately model the increasing protein stability observed at high cocktail loadings, the model was modified to include the presence of additives in the cocktail (R2 = 0.98). By combining the measurement of total protein concentration with the proposed Novozyme 188 protein aggregation model, the endo- and exoglucanases concentration in the solid and liquid phases during hydrolysis can be more accurately determined. This methodology can be applied to various systems leading to optimization of enzyme loading by minimizing the excess of endo- and exoglucanases. In addition, the monitoring of endo- and exoglucanases concentrations can be used to build mass balances of enzyme recycling processes and to techno-economically evaluate the viability of enzyme recycling

Characterization of Xylan Utilization and Discovery of a New Endoxylanase in Thermoanaerobacterium Saccharolyticum through Targeted Gene Deletions

The economical production of fuels and commodity chemicals from lignocellulose requires the utilization of both the cellulose and hemicellulose fractions. Xylanase enzymes allow greater utilization of hemicellulose while also increasing cellulose hydrolysis. Recent metabolic engineering efforts have resulted in a strain of Thermoanaerobacterium saccharolyticum that can convert C5 and C6 sugars, as well as insoluble xylan, into ethanol at high yield. To better understand the process of xylan solubilization in this organism, a series of targeted deletions were constructed in the homoethanologenic T. saccharolyticum strain M0355 to characterize xylan hydrolysis and xylose utilization in this organism. While the deletion of -xylosidase xylD slowed the growth of T. saccharolyticum on birchwood xylan and led to an accumulation of short-chain xylo-oligomers, no other single deletion, including the deletion of the previously characterized endoxylanase XynA, had a phenotype distinct from that of the wild type.This result indicates a multiplicity of xylanase enzymes which facilitate xylan degradation in T. saccharolyticum. Growth on xylan was prevented only when a previously uncharacterized endoxylanase encoded by xynC was also deleted in conjunction with xynA. Sequence analysis of xynC indicates that this enzyme, a low-molecular-weight endoxylanase with homology to glycoside hydrolase family 11 enzymes, is secreted yet untethered to the cell wall. Together, these observations expand our understanding of the enzymatic basis of xylan hydrolysis by T. saccharolyticum

The fate of lignin during hydrothermal pretreatment

Background: Effective enzymatic hydrolysis of lignocellulosic biomass benefits from lignin removal, relocation, and/or modification during hydrothermal pretreatment. Phase transition, depolymerization/repolymerization, and solubility effects may all influence these lignin changes. To better understand how lignin is altered, Populus trichocarpa x P. deltoides wood samples and cellulolytic enzyme lignin (CEL) isolated from P. trichocarpa x P. deltoides were subjected to batch and flowthrough pretreatments. The residual solids and liquid hydrolysate were characterized by gel permeation chromatography, heteronuclear single quantum coherence NMR, compositional analysis, and gas chromatography–mass spectrometry. Results Changes in the structure of the solids recovered after the pretreatment of CEL and the production of aromatic monomers point strongly to depolymerization and condensation being primary mechanisms for lignin extraction and redeposition. The differences in lignin removal and phenolic compound production from native P. trichocarpa x P. deltoides and CEL suggested that lignin-carbohydrate interactions increased lignin extraction and the extractability of syringyl groups relative to guaiacyl groups. Conclusions These insights into delignification during hydrothermal pretreatment point to desirable pretreatment strategies and plant modifications. Because depolymerization followed by repolymerization appears to be the dominant mode of lignin modification, limiting the residence time of depolymerized lignin moieties in the bulk liquid phase should reduce lignin content in pretreated biomass. In addition, the increase in lignin removal in the presence of polysaccharides suggests that increasing lignin-carbohydrate cross-links in biomass would increase delignification during pretreatment.Applied Science, Faculty ofChemical and Biological Engineering, Department ofNon UBCReviewedFacult

Kraft Pulping of Softwood Chips with Mild Hot Water Pre-hydrolysis to Understand the Effects of Wood Chip Thickness

Hemicelluloses consume alkali during kraft pulping and dissolve in the black liquor as a low energy fuel. Acidic pre-hydrolysis of softwood chips removes hemicelluloses but preserves cellulose content prior to pulping. This study compared mild pre-hydrolysis (140 °C) kraft pulping with conventional kraft pulping of commercial softwood chips at two H-factors for wood chips with thickness ranging from less than 2 mm to over 6 mm. The chip thickness less than 2 mm increased hemicelluloses oligomer yield and showed little influence on pulp fiber yield. However, the kraft pulp fiber length decreased 5.6%. Kappa number and fiber reject increased dramatically when chip thickness was greater than 6 mm. The detailed compositional analysis of kraft pulp and fiber quality analysis indicate that pre-hydrolysis followed by kraft pulping enhanced delignification with limited reduction of fiber length and width, and increased kinks. Strategic considerations for the integration of pre-hydrolysis into kraft pulping for future biorefineries were outlined

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

Lignin depolymerization and monomeric evolution during fast pyrolysis oil upgrading with hydrogen from glycerol aqueous phase reforming

A novel approach to Fast Pyrolysis Oil (FPO) upgrading with hydrogen from glycerol aqueous phase reforming (APR) was conducted in a biphasic solution. FPO contains both monomer and polymer compounds which rich in oxygen, giving high acidity and low stability. Hydrogen demanding reaction of depolymerization and hydrodeoxygenation (HDO), often called upgrading, is required to improve FPO properties by converting these compounds to hydrocarbon monomers. APR reaction of glycerol where glycerol is reacted with water to produce hydrogen is one of the renewable choices to obtain hydrogen. Prior to upgrading of FPO, catalyst screening and reaction optimization were studied using phenol as a model compound. Upgrading of FPO with in situ glycerol APR was conducted with Pt/C, facilitating hydrogen production (APR) and utilization (hydrogenation), and H-ZSM-5, facilitating dehydration reaction. n-Decane was added to the reaction as a co-solvent to prevent the condensation of the non-polar fragments of FPO which led to coke formation. Upon upgrading the weight average molecular weight (Mw), polydispersity index (PDI), and oxygen to carbon (O/C) ratio of FPO decreased. The highest hydrocarbon yield (7.7 FPO basis or 34.6 lignin basis) was obtained by combining Pt/C and H-ZSM-5 catalysts with n-decane as a co-solvent. Evidence of progressive depolymerization and sequential demethoxylation, hydrogenation, and deoxygenation during upgrading were observed in the products. © 2022 Elsevier Lt

Enzyme Recycling by Adsorption during Hydrolysis of Oxygen-Delignified Wheat Straw

Enzyme recycling by adsorption from supernatant to fresh substrate is a promising strategy to reduce enzyme expenses and the production cost of lignocellulosic ethanol. The study was performed using oxygen-delignified wheat straw, and the effect of lignin content, enzyme loading, and hydrolysis time on recycling was determined. The percent of recycled cellulases, 0–35% of initial cellulase loading, increased with increasing enzyme loading and hydrolysis time but decreased with increasing lignin content. Cellulose conversions of 10–71% were achieved during the second hydrolysis round using only recycled cellulases indicating the existence of a highly active subset of enzymes. To achieve constant production of sugars during enzyme recycling, fresh cellulases were loaded before the second hydrolysis round to match the cellulase loading used in the first round. Subsequently, similar glucose, xylose, and protein concentrations were obtained at the end of the first and second rounds for all conditions. Recycling mass balances were developed to support future techno-economic analyses to determine the impact of enzyme recycling on the cost of ethanol

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