Cellulase retention and sugar removal by membrane ultrafiltration during lignocellulosic biomass hydrolysis

Technologies suitable for the separation and reuse of cellulase enzymes during the enzymatic saccharification of pretreated corn stover are investigated to examine the economic and technical viability of processes that promote cellulase reuse while removing inhibitory reaction products such as glucose and cellobiose. The simplest and most suitable separation is a filter with relatively large pores on the order of 20–25 mm that retains residual corn stover solids while passing reaction products such as glucose and cellobiose to form a sugar stream for a variety of end uses. Such a simple separation is effective because cellulase remains bound to the residual solids. Ultrafiltration using 50-kDa polyethersulfone membranes to recover cellulase enzymes in solution was shown not to enhance further the saccharification rate or overall conversion. Instead, it appears that the necessary cellulase enzymes, including β-glucosidase, are tightly bound to the substrate; when fresh corn stover is contacted with highly washed residual solids, without the addition of fresh enzymes, glucose is generated at a high rate. When filtration was applied multiple times, the concentration of inhibitory reaction products such as glucose and cellobiose was reduced from 70 to 10 g/L. However, an enhanced saccharification performance was not observed, most likely because the concentration of the inhibitory products remained too high. Further reduction in the product concentration was not investigated, because it would make the reaction unnecessarily complex and result in a product stream that is much too dilute to be useful. Finally, an economic analysis shows that reuse of cellulase can reduce glucose production costs, especially when the enzyme price is high. The most economic performance is shown to occur when the cellulase enzyme is reused and a small amount of fresh enzyme is added after each separation step to replace lost or deactivated enzyme.     

A comparative study on chemical conversion of cellulose between the batch-type and flow-type systems in supercritical water

Microcrystalline cellulose (avicel) was treated in supercritical waterusing batch-type and flow-type systems. The flow-type system made it possibletoshorten the heating, treating and cooling times, compared with the batch-typesystem. As a result, the flow-type system was able to liquefy avicel withoutproducing any supercritical water-insoluble residue. Although hydrolyzedproducts such as glucose and fructose, and pyrolyzed products such aslevoglucosan, 5-hydroxymethyl furfural, erythrose, methylglyoxal,glycolaldehydeand dihydroxyacetone were found in common from the water-soluble portiontreatedby both systems, the flow-type system gave a water-soluble portion with morehydrolyzed and less pyrolyzed products, together with water-solubleoligosaccharides consisting of cellobiose to cellododecaose and theirdecomposedproducts at their reducing end of glucose, such as[–glucopyranosyl]_1–11 –levoglucosan,[–glucopyranosyl]_1–11 –erythrose and[–glucopyranosyl]_1–11 –glycolaldehyde. Inaddition, the precipitates of polysaccharides were recovered after 12h setting of the water-soluble portion. These results indicatedthat the flow-type system can hydrolyze cellulose with minimizing pyrolyzedproducts. On the other hand, the batch-type system resulted in a higher yieldof the pyrolyzed products due to the longer treatment, but a higher yield ofglucose due possibly to the higher pressure and concomitantly higher ionicproduct of water. Based on these lines of evidence, the process to increase theyield of the sugar is discussed under supercritical water treatment.     

Saccharification and alcohol fermentation in starch solution of steam-exploded potato

Steam explosion pretreatment of potato for the efficient production of alcohol was experimentally studied. The amount of water-soluble starch increased with the increase of steam pressure, but the amounts of methanol-soluble material and Klason lignin remained insignificant, regardless of steam pressure. The potatoes exploded at high pressure were hydrolyzed into a low molecular liquid starch, and then easily converted into ethanol by simultaneous saccharification and fermentation using mixed microorganisms: an amylolytic microorganism,Aspergillus awamori, and a fermentation microorganism,Saccharomyces cerevisiae. The maximal ethanol concentration was 4.2 g/L in a batch culture at 15 g/L starch concentration, and 3.6 g/L in a continuous culture fed the same starch concentration. In the fed-batch culture, the maximal ethanol concentration increased more than twofold, compared to the batch culture.     

Cellulose hydrolysis – the role of monocomponent cellulases in crystalline cellulose degradation

Changes in the molecular structure of cellulose during hydrolysis with four recombinant -1,4-glycanases from the cellulolytic bacterium Cellulomonas fimi were assessed and compared in an attempt to elucidate the mechanism of crystalline cellulose degradation. It was apparent that the two endoglucanases, Cel6A and Cel5A, degraded sigmacell cellulose differently; Cel5A liberated more soluble sugars (cellobiose and cellotriose) and significantly altered the molecular weight distribution, while Cel6A had a limited effect on the polymer size and liberated primarily cellobiose and glucose. Additionally, both endoglucanases slightly increased the crystallinity of cellulose. In contrast, the cellobiohydrolases, Cel6B and Cel48A, had no effect on cellulose molecular weight and liberated only cellobiose and cellotriose. However, Cel48A was shown to be effective at reducing the crystallinity of the cellulosic substrate, while Cel6B increased the crystallinity index. Synergistic hydrolysis using combinations of the different enzymes showed that, although the cellulose was extensively hydrolysed, the molecular structure of the substrate was similar to the original material. This phenomenon suggests that the actions of individual monocomponent enzymes are offset by the concurrent modification by the complementing enzymes during synergistic hydrolysis.     

Analysis of biomass cellulose in simultaneous saccharification and fermentation processes

A direct method for determining the cellulose content of biomass residues resulting from simultaneous saccharifiaction and fermentation (SSF) experiment has been developed and evaluated. The method improves on classical cellulose assays by incorporating the enzymatic removal of yeast glucans from the biomass residue prior to acid hydrolysis and subsequent quantification of cellulose-derived glucose. An appropriate cellulasefree, commercially available, yeast-lysing enzyme preparation fromCytophaga was identified. A freeze-drying step was identified as necessary to render the SSF yeast cells susceptible to enzymatic lysis. The method was applied to the analysis of cellulose and yeast-associated glucans in SSF residues from three pretreated feedstocks; hybrid poplar, switchgrass, and cornstover. Cellulose assays employing the lysing-enzyme preparation demonstrated relative errors up to 7.2% when yeast-associated glucans were not removed prior to analysis of SSF residues. Enzymatic lysis of SSF yeast cells may be viewed as a general preparatory procedure to be used prior to subsequent chemical and physical analysis of SSF residues. Oregon State University Agricultural Experiment Station Technical Publication Number 10977.     

高分子添加剂对纤维素酶糖化力的影响

研究了几种高分子材料对纤维素酶水解作用的影响,强聚阳离子(PⅠ、PⅡ)、中强聚阴离子(PⅢ)高分子材料能促进酶的糖化作用,而强聚阴离子(PⅣ)高分子材料则不能,添加PⅠ(1.3×10~(-1)mol/L)、PⅡ(1.3×10~3mol/L)、PⅢ(1.3×10~2mol/L)分别使纤维素酶棉花糖化力提高190％、45％、75％,初步探讨了高分子材料促进纤维素酶糖化力的机理。     

Research on the Conditions of Enzymatic Saccharification for Sugarcane Bagasse

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Enhanced saccharification of lignocellulosic biomass with1-allyl-3-methylimidazolium chloride（Amim Cl） pretreatment

A simple and efficient method of enhancing biomass saccharification by microwave-assisted pretreatment with dimethyl sulfoxide/1-allyl-3-methylimidazolium chloride is proposed. Softwood（pine wood（PW））, hardwoods（poplar wood, catalpa bungi, and Chinese parasol）, and agricultural wastes（rice straw, wheat straw, and corn stover（CS）） were exploited. Results showed that the best pretreatment effect was in PW with 54.3% and 31.7% dissolution and extraction ratios, respectively. The crystal form of cellulose in PW extract transformed from I to II, and the contended cellulose ratio and glucose conversion ratio reached 85.1% and 85.4%, respectively. CS after steam explosion achieved a similar pretreating effect as PW, with its cellulose hydrolysis ratio reaching as high as 91.5% after IL pretreatment.     

Simultaneous saccharification and fermentation of lignocellulose

Simultaneous saccharification and fermentation (SSF) processes for producing ethanol from lignocellulose are capable of improved hydrolysis rates, yields, and product concentrations compared to separate hydrolysis and fermentation (SHF) systems, because the continuous removal of the sugars by the yeasts reduces the end-product inhibition of the enzyme complex. Recent experiments using Genencor 150L cellulase and mixed yeast cultures have produced yields and concentrations of ethanol from cellulose of 80% and 4.5%, respectively. The mixed culture was employed because B.clausenii has the ability to ferment cellobiose (further reducing end-product inhibition), while the brewing yeastS. cerevisiae provides a robust ability to ferment the monomeric sugars. These experimental results are combined with a process model to evaluate the economics of the process and to investigate the effect of alternative processes, conditions, and organisms.     

Overview of Special Session B—Compositional and Structural Analysis of Biomass

Special Session B at the 29th Symposium on Biotechnology for Fuels and Chemicals was the first invited session at this symposium devoted to analytical methods. The special topic was added in response to numerous requests for information on new and innovative methods that could be applied in the growing renewable fuels industry. Presentation topics include analytical methods for the characterization and analysis of maize traits, tools for investigating cell wall limitations to enzymatic degradation, methods for customizing enzyme cocktails for biomass, new techniques for the analysis of carbohydrates, analytical methods that enhance our understanding of pretreatment, improved methods for monitoring process intermediates, and published standard analytical methods for biomass conversion processes.